BackgroundBed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance.Methodology and Principal FindingsUsing 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database.ConclusionsTo our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies.

BackgroundThe insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level.Methodology and Principal FindingsNewer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level.Conclusions and SignificanceTo our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis.

Background The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. Results We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. Conclusions Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

The Emerald ash borer (EAB, Agrilus planipennis Fairmaire) an exotic invasive insect pest has killed millions of ash trees (Fraxinus spp.) across North America and threatens billions more. We validated six A. planipennis reference genes (actin, ACT; beta tubulin, β‐TUB; glyceraldehyde‐3‐phosphate dehydrogenase, GAPDH; ribosomal protein, RPL7; translation elongation factor 1α, TEF‐1α; and ubiquitin, UBQ) using geNorm, Normfinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies. The stability of the six reference genes was evaluated in different larval tissues, developmental stages and two treatments of A. planipennis using quantitative real‐time polymerase chain reaction. Although there was no consistent ranking observed among the reference genes across the samples, the overall analysis revealed TEF‐1α as the most stable reference gene. GAPDH and ACT showed least stability for all the samples studied. We conclude that TEF‐1α is the most appropriate reference gene for gene expression studies in A. planipennis. Results obtained can be applicable for transcript profiling in other invasive insect pests. Further, these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post‐validation.

BackgroundSecond generation lignocellulosic feedstocks are being considered as an alternative to first generation biofuels that are derived from grain starches and sugars. However, the current pre-treatment methods for second generation biofuel production are inefficient and expensive due to the recalcitrant nature of lignocellulose. In this study, we used the lower termite Reticulitermes flavipes (Kollar), as a model to identify potential pretreatment genes/enzymes specifically adapted for use against agricultural feedstocks.ResultsMetatranscriptomic profiling was performed on worker termite guts after feeding on corn stover (CS), soybean residue (SR), or 98% pure cellulose (paper) to identify (i) microbial community, (ii) pathway level and (iii) gene-level responses. Microbial community profiles after CS and SR feeding were different from the paper feeding profile, and protist symbiont abundance decreased significantly in termites feeding on SR and CS relative to paper. Functional profiles after CS feeding were similar to paper and SR; whereas paper and SR showed different profiles. Amino acid and carbohydrate metabolism pathways were downregulated in termites feeding on SR relative to paper and CS. Gene expression analyses showed more significant down regulation of genes after SR feeding relative to paper and CS. Stereotypical lignocellulase genes/enzymes were not differentially expressed, but rather were among the most abundant/constitutively-expressed genes.ConclusionsThese results suggest that the effect of CS and SR feeding on termite gut lignocellulase composition is minimal and thus, the most abundantly expressed enzymes appear to encode the best candidate catalysts for use in saccharification of these and related second-generation feedstocks. Further, based on these findings we hypothesize that the most abundantly expressed lignocellulases, rather than those that are differentially expressed have the best potential as pretreatment enzymes for CS and SR feedstocks.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1502-8) contains supplementary material, which is available to authorized users.

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54Background: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally 55 invasive pest and plant virus vector on a wide array of food, fiber and ornamental crops. While 56 there are numerous studies centered on thrips pest and vector biology, feeding behaviors, ecology, 57 and insecticide resistance, the underlying genetic mechanisms of the processes governing these 58 areas of research are largely unknown. To address this gap, we present the F. occidentalis draft 59 genome assembly and official gene set. 60Results: We report on the first genome sequence for any member of the insect order Thysanoptera. 61 Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly 62 (size = 415.8 Mb, scaffold N50 = 948.9 Kb) revealed a relatively complete and well-annotated 63 assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) 64 compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, 65 of which ~10% were manually verified and corrected by our consortium. We focused on manual 66 annotation, phylogenetic and expression evidence analyses for gene sets centered on primary 67 themes in the life histories and activities of plant-colonizing insects. Highlights include: 1) 68 divergent clades and large expansions in genes associated with environmental sensing 69 (chemosensory receptors) and detoxification (CYP4, CYP6 and CCE enzymes) of substances 70 encountered in agricultural environments; 2) a comprehensive set of salivary gland-associated 71 genes supported by enriched expression; 3) apparent absence of members of the IMD innate 72 3 immune defense pathway; and 4) developmental-and sex-specific expression analyses of genes 73 associated with progression from larvae to adulthood through neometaboly, a distinct form of 74 maturation compared to complete metamorphosis in the Holometabola. 75 Conclusions: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior 76 of this insect pest to find, colonize and survive on a widely diverse array of plants. The genomic 77 resources presented here enable a more complete analysis of insect evolution and biology, 78 providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers 79 a genomic benchmark for molecular and evolutionary investigations of other thysanopteran 80 species. 81 KEY WORDS 82 Thysanoptera, western flower thrips, hemipteroid assemblage, insect genomics, tospovirus, 83 virus-vector interactions, salivary glands, chemosensory receptors, opsins, detoxification, innate 84 immunity 85 86 BACKGROUND 87Thrips are small, polyphagous and cosmopolitan insects that comprise the order Thysanoptera. 88Thysanoptera lies within the Paraneoptera, also commonly called the "hemipteroid assemblage" 89 which also includes the orders Hemiptera, Psocoptera, and Phthiraptera. Among the over 7,000 90 reported thrips species classified into nine families with an additional five identified from fossil 91 species [1]...