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Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions (basement membrane, BM and extracellular matrix proteins, ECM), and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV- infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

La transmission des virus de plantes par vecteurs arthropodes s’opère via des interactions spécifiques entre plante, virus et vecteur. Les stratégies de transmission par vecteur sont déterminées par : la localisation du virus transmissible dans l’arthropode (transmission par virus circulant ou non circulant dans le vecteur), la durée de la relation virusvecteur (non persistante, semi-persistante ou persistante) et s’il y a réplication ou pas du virus dans l’insecte (tranmission propagative ou non propagative). La plupart des virus de plantes sont transmis horizontalement par les arthropodes, même si des réovirus, rhabodovirus et tenuivirus peuvent aussi être transmis verticalement chez le vecteur. Ce chapitre analyse des progrès récents et des questions de longue date de la biologie des vecteurs.

Insect salivary glands play an important role for host feeding, specifically by secreting salivary proteins for digestion and potentially modulating host defenses. Compared to other hemipterans, the significance of salivary glands is less studied in the black-faced leafhopper, Graminella nigrifrons, a crop pest that vectors several agronomically important plant viruses. To identify functionally important genes in the salivary glands of the black-faced leafhopper, we compared transcriptomes between adult salivary glands (SG) and the remaining carcasses. We identified 14,297 salivary gland-enriched transcripts and 195 predicted secretory peptides (i.e., with a signal peptide and extracellular localization characteristics). Overall, the SG transcriptome included functions such as &lsquo, oxidoreduction&rsquo, &lsquo, membrane transport&rsquo, and &lsquo, ATP-binding&rsquo, which might be important for the fundamental physiology of this tissue. We further evaluated transcripts with potential contributions in host feeding using RT-qPCR. Two SG-enriched transcripts (log2 fold change &gt, 5), GnP19 and GnE63 (a putative calcium binding protein), were significantly upregulated in maize-fed adults relative to starved adults, validating their importance in feeding. The SG-enriched transcripts of the black-faced leafhopper could play a potential role for interacting with maize and could be targets of interest for further functional studies and improve pest control and disease transmission.

This study investigated saccharification and protein-level responses to the candidate biofuel feedstocks corn stover (CS) and soybean residue (SR) by the gut of a lower termite. The focus termite in this work was Reticulitermes flavipes, which is a highly efficient lignocellulose digester that houses a mixture of prokaryotic and eukaryotic microbes in its gut. Our specific objectives were to (i) measure saccharification potential of the CS and SR feedstocks by termite gut protein extracts, (ii) identify specific proteins in the termite gut responding to feeding on CS and SR diets, and (iii) evaluate gut lignocellulase and accessory enzyme activity responses to CS and SR feeding. Cellulose paper was the control diet. Although CS was saccharified at higher levels, termite gut protein extracts saccharified both CS and SR irrespective of feedstock loading. Consumption of the CS and SR feedstocks by termites resulted in surprisingly few differences in gut protein profiles, with the main exception being elevated myosin abundance with SR feeding. Activity of potential lignocellulases and accessory enzymes was generally similar between CS and SR fed guts as well; however, cellobiohydrolase/exoglucanase activity was higher with CS feeding and glutathione peroxidase activity with SR feeding. These findings have significance from two perspectives. First, SR feeding / digestion appears to cause physiological stress in the termite gut that likely would extend to other types of microbial environments including those within industrial bioreactors. Second, because termites can survive on exclusive CS and SR diets and their guts exhibit clear CS and SR saccharification activity, this validates the R. flavipes system as a potential source for CS and SR degrading enzymes; in particular, cellobiohydrolases/exoglucanases and glutathione peroxidases from this system appear to play roles in CS and SR breakdown.

The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-μ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-μ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles.

Recent advances of Piwi-interacting RNAs (piRNAs) have enabled a better understanding of their putative roles in the protection of germ cells against selfish genetic elements. The germ plasm proteins namely, PIWI and VASA, are key players in regulation of piRNAs. However, the biogenesis of piRNAs remains poorly understood. We report the characterization and the transcript profiles of a PIWI and VASA gene in various tissues and developmental stages of the emerald ash borer (Agrilus planipennis), an exotic invasive insect pest that has attained a high impact status worldwide. The deduced amino acid sequences of both these genes (herein referred to “ApPIWI and ApVASA”) revealed homology with similar proteins from other insect and non-insect taxa. Peak transcript levels for ApPIWI and ApVASA were observed in the ovaries (compared to the other tissues assayed) and in adults (compared to the other developmental stages assayed). To our knowledge this is the first report of profiling piRNA pathway genes in A. planipennis. These results provide new insights into the putative function of the PIWI and VASA proteins in insects.

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.

Background Second 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. Results Metatranscriptomic 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. Conclusions These 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 material The online version of this article (doi:10.1186/s12864-015-1502-8) contains supplementary material, which is available to authorized users.

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; α-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.

Phytophagous insects frequently encounter reactive oxygen species (ROS) from exogenous and endogenous sources. To overcome the effect of ROS, insects have evolved a suite of antioxidant defense genes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). The emerald ash borer ( Agrilus planipennis Fairmaire), an exotic invasive insect pest from Asia has killed millions of ash trees and continues to invade North America at a rapid pace. From an on-going expressed sequence tag (EST) project of A. planipennis larval tissues, we identified ESTs coding for a Cu–Zn SOD ( Ap SOD1), a CAT ( Ap CAT1) and a GPX ( Ap GPX1). A multiple sequence alignment of the derived A. planipennis sequences revealed high homology with other insect sequences at the amino acid level. Phylogenetic analysis of Ap SOD1 grouped it with Cu–Zn SODs of other insect taxa. Quantitative real time PCR (qRT-PCR) analysis in different larval tissues (midgut, fat body, Malpighian tubule and cuticle) revealed high mRNA levels of Ap CAT1 in the midgut. Interestingly, high mRNA levels for both Ap SOD1 and Ap GPX1 were observed in the Malpighian tubules. Assay of mRNA levels in developmental stages (larva, prepupa and adults) by qRT-PCR indicated high transcript levels of Ap CAT1 and Ap GPX1 in larval and prepupal stages with a decline in adults. On the other hand, the transcript levels of Ap SOD1 were observed to be constitutive in all the developmental stages assayed. Results obtained reflect a plausible role of these A. planipennis antioxidant genes in quenching ROS from both diet (ash allelochemicals) as well as endogenous sources. These studies further help in understanding the adaptation/invasiveness of A. planipennis .

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.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.