Salivary Gland Transcriptome Research Articles

Exploring the contribution of the salivary gland and midgut to digestion in the swede midge (Contarinia nasturtii) through a genomics-guided approach.

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and β-fructofuranosidases (invertases) were identified. Genes encoding β-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.

Salivary gland transcriptome analysis in Hirudinaria sp. during different feeding periods and functional studies of HspHirudin

Salivary gland transcriptome analysis in Hirudinaria sp. during different feeding periods and functional studies of HspHirudin

Identification of salivary proteins of the cowpea aphid Aphis craccivora by transcriptome and LC-MS/MS analyses

Aphid salivary proteins mediate the interaction between aphids and their host plants. Moreover, these proteins facilitate digestion, detoxification of secondary metabolites, as well as activation and suppression of plant defenses. The cowpea aphid, Aphis craccivora, is an important sucking pest of leguminous crops worldwide. Although aphid saliva plays an important role in aphid plant interactions, knowledge of the cowpea aphid salivary proteins is limited. In this study, we performed transcriptomic and LC-MS/MS analyses to identify the proteins present in the salivary glands and saliva of A. craccivora. A total of 1,08,275 assembled transcripts were identified in the salivary glands of aphids. Of all these assembled transcripts, 53,714 (49.11%) and 53,577 (49.48%) transcripts showed high similarity to known proteins in the Nr and UniProt databases, respectively. A total of 2159 proteins were predicted as secretory proteins from the salivary gland transcriptome dataset, which contain digestive enzymes, detoxification enzymes, previously known effectors and elicitors, and potential proteins whose functions have yet to be determined. The proteomic analysis of aphid saliva resulted in the identification of 171 proteins. Tissue-specific expression of selected genes using RT-PCR showed that three genes were expressed only in the salivary glands. Overall, our results provide a comprehensive repertoire of cowpea aphid salivary proteins from the salivary gland and saliva, which will be a good resource for future effector functional studies and might also be useful for sustainable aphid management.

Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are a leading cause of morbidity and mortality, which are compounded by drug resistance. By manipulating the coagulation system, S. aureus gains a significant advantage over host defense mechanisms, with hypercoagulation induced by S. aureus potentially aggravating infectious diseases. Recently, we and other researchers identified that a higher level of LL-37, one endogenous antimicrobial peptide with a significant killing effect on S. aureus infection, resulted in thrombosis formation through the induction of platelet activation and potentiation of the coagulation factor enzymatic activity. In the current study, we identified a novel antimicrobial peptide (RK22) from the salivary gland transcriptome of Hirudinaria manillensis (H. manillensis) through bioinformatic analysis, and then synthesized it, which exhibited good antimicrobial activity against S. aureus, including a clinically resistant strain with a minimal inhibitory concentration (MIC) of 6.25 μg/mL. The RK22 peptide rapidly killed S. aureus by inhibiting biofilm formation and promoting biofilm eradication, with good plasma stability, negligible cytotoxicity, minimal hemolytic activity, and no significant promotion of the coagulation system. Notably, administration of RK22 significantly inhibited S. aureus infection and the clinically resistant strain in vivo. Thus, these findings highlight the potential of RK22 as an ideal treatment candidate against S. aureus infection.

Salivary protein expression profiles of five species of Pentatomidae (Hemiptera)

Abstract Stink bug (Hemiptera: Pentatomidae) development typically requires feeding on a diversity of plant species and various plant tissues. During feeding, stink bugs discharge salivary enzymes with roles in extraoral digestion and countering plant defense responses. Although previous research has described digestive salivary proteins from stink bugs, less is known of the salivary proteins involved in the suppression of plant defenses. We sequenced the transcriptomes of salivary glands dissected from five stink bug species collected from non-crop habitats in Washington: Halyomorpha halys (Stål), Nezara viridula L., Euschistus conspersus (Uhler), Thyanta pallidovirens (Stål), and Chlorochroa ligata (Say). We identified a total of 677 candidate secreted proteins from the salivary glands of the five species. Based on work from other insects, many of the proteins have potential functions in the suppression of plant defense signaling and deactivation of plant defense molecules. We also identified salivary proteins with potential roles in the extraoral digestion of plant tissues, protection from entomopathogens, and deposition of salivary sheaths. This report provides a curation of putative salivary effector genes for further functional analysis.

Function-guided selection of salivary antigens from Ornithodoros erraticus argasid ticks and assessment of their protective efficacy in rabbits

The identification of new protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that have key biological functions. Bioactive proteins playing key functions for tick feeding and pathogen transmission are secreted into the host via tick saliva. Adult argasid ticks must resynthesise and replace these proteins after each feeding to be able to repeat new trophogonic cycles. Therefore, these proteins are considered interesting antigen targets for tick vaccine development. In this study, the salivary gland transcriptome and saliva proteome of Ornithodoros erraticus females were inspected to select and test new vaccine candidate antigens. For this, we focused on transcripts overexpressed after feeding that encoded secretory proteins predicted to be immunogenic and annotated with functions related to blood ingestion and modulation of the host defensive response. Completeness of the transcript sequence, as well as a high expression level and a high fold-change after feeding were also scored resulting in the selection of four candidates, an acid tail salivary protein (OeATSP), a multiple coagulation factor deficiency protein 2 homolog (OeMCFD2), a Cu/Zn-superoxide dismutase (OeSOD) and a sulfotransferase (OeSULT), which were later produced as recombinant proteins. Vaccination of rabbits with each individual recombinant antigen induced strong humoral responses that reduced blood feeding and female reproduction, providing, respectively, 46.8%, 45.7%, 54.3% and 31.9% protection against O. erraticus infestations and 0.7%, 3.9%, 3.1% and 8.7% cross-protection against infestations by the African tick, Ornithodoros moubata. The joint protective efficacy of these antigens was tested in a second vaccine trial reaching 58.3% protection against O. erraticus and 18.6% cross-protection against O. moubata. These results (i) provide four new protective salivary antigens from argasid ticks that might be included in multi-antigenic vaccines designed for the control of multiple tick species; (ii) reveal four functional protein families never tested before as a source of protective antigens in ticks; and (iii) show that multi-antigenic vaccines increase vaccine efficacy compared with individual antigens. Finally, our data add value to the salivary glands as a protective antigen source in argasids for the control of tick infestations.

A comparison of Illumina and PacBio methods to build tick salivary gland transcriptomes confirms large expression of lipocalins and other salivary protein families that are not represented in available tick genomes

Tick saliva helps blood feeding by its antihemostatic and immunomodulatory activities. Tick salivary gland transcriptomes (sialotranscriptomes) revealed thousands of transcripts coding for putative secreted polypeptides. Hundreds of these transcripts code for groups of similar proteins, constituting protein families, such as the lipocalins and metalloproteases. However, while many of these transcriptome-derived protein sequences matches sequences predicted by tick genome assemblies, the majority are not represented in these proteomes. The diversity of these transcriptome-derived transcripts could derive from artifacts generated during assembly of short Illumina reads or derive from polymorphisms of the genes coding for these proteins. To investigate this discrepancy, we collected salivary glands from blood-feeding ticks and, from the same homogenate, made and sequenced libraries following Illumina and PacBio protocols, with the assumption that the longer PacBio reads would reveal the sequences generated by the assembly of Illumina reads. Using both Rhipicephalus zambeziensis and Ixodes scapularis ticks, we have obtained more lipocalin transcripts from the Illumina library than the PacBio library. To verify whether these unique Illumina transcripts were real, we selected 9 uniquely Illumina-derived lipocalin transcripts from I. scapularis and attempted to obtain PCR products. These were obtained and their sequences confirmed the presence of these transcripts in the I. scapularis salivary homogenate. We further compared the predicted salivary lipocalins and metalloproteases from I. scapularis sialotranscriptomes with those found in the predicted proteomes of 3 publicly available genomes of I. scapularis. Results indicate that the discrepancy between the genome and transcriptome sequences for these salivary protein families is due to a high degree of polymorphism within these genes.

Saliva proteome of partially- and fully-engorged adult female Haemaphysalis flava ticks

Tick saliva is a reservoir of bioactive proteins. Saliva protein compositions change dynamically during blood-feeding. Decipherment of protein profiles in different blood-feeding stages may bring deeper insight into tick feeding physiology and provide targets for immunologic control alternatives. However, having the infancy of tick genome sequencing, assembly, annotation, and limited knowledge of tick salivary proteins restrain the data interpretation. Here, we aimed to depict the saliva protein profile in partially- (PE) and fully-engorged (FE) Haemaphysalis flava ticks, with a special focus on the analysis of those uncharacterized proteins. Saliva was collected from PE and FE adult female H. flava ticks. Saliva proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS). MS data were searched against an in-house salivary gland transcriptome library for identification of tick-derived proteins. Abundances of proteins were compared between PE and FE ticks. The uncharacterized proteins detected in saliva were further bioinformatically analyzed. In total, 614 proteins were identified including 94 host proteins and 520 tick-derived proteins. The 226 tick-derived high-confidence proteins were classified into 10 categories: transporters, enzymes, protease inhibitors, immunity-related proteins, lipocalins, glycine-rich proteins, muscle proteins, secreted proteins, uncharacterized proteins and others. A total of 98 proteins were shared in both PE and FE with 74 only in PE and 54 only in FE. Abundances of 24 shared proteins were significantly higher in PE. The profile of top 15 most abundant proteins was also different between PE and FE ticks. The 65 uncharacterized proteins detected in tick saliva were branched into subclusters 1 A, 1B, 2, 3 A, 3B and 3 C based on particular motifs like RGD, LRR, indicating their diverse predicted functions like anti-coagulation, regulation of innate immune, or other functions. This study provides and compares saliva proteomes of H. flava ticks in two feeding stages with special cluster analysis on the uncharacterized proteins. Further investigations are needed to confirm the roles of these uncharacterized proteins in ticks.

Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia

Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin “core” motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China’s growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs.

Transcriptomic and proteomic analyses provide insights into host adaptation of a bamboo-feeding aphid.

Salivary glands and their secreted proteins play an important role in the feeding process of sap-sucking aphids. The determination of saliva composition is an important step in understanding host plant adaptation of aphids. Pseudoregma bambucicola is a severe bamboo pest in subtropical areas and the only aphid species that can exclusively feed on hard stalks of bamboos. How this species can penetrate and degrade hard bamboo cell walls and utilize a very specialized niche are important unanswered questions. In this study, comprehensive analyses based on transcriptome sequencing, RT-qPCR, liquid chromatography-tandem spectrometry (LC-MS/MS) and bioinformatics were conducted on dissected salivary glands and secreted saliva of P. bambucicola to characterize the overall gene expression and salivary protein composition, and to identify putative effector proteins important for aphid-plant interactions. Some secretory proteins homologous to known aphid effectors important for aphid-plant interactions, such as digestive enzymes, detoxifying and antioxidant enzymes and some effectors modulating plant defenses, are also detected in salivary gland transcriptome and salivary gland and/or saliva secretomes in P. bambucicola. This indicates that these effectors are probably be essential for enabling P. bambucicola feeding on bamboo host. Although several plant cell wall degrading enzymes (PCWDEs) can be identified from transcriptome, most of the enzymes identified in salivary glands showed low expression levels and they only represent a small fraction of the complete set of enzymes for degrading cellulose and hemicellulose. In addition, our data show that P. bambucicola has no its own ability to produce pectinases. Overall, our analyses indicate that P. bambucicola may lose its own ability to express and secrete key PCWDEs, and its adaptation to unique feeding habit may depend on its symbiotic bacteria.

Characterization of the swede midge, Contarinia nasturtii, first instar larval salivary gland transcriptome

Proteins in saliva of gall-forming insect larvae govern insect-host plant interactions. Contarinia nasturtii, the swede midge, is a pest of brassicaceous vegetables (cabbage, cauliflower, broccoli) and canola. We examined the salivary gland (SG) transcriptome of first instar larvae reared on Brassica napus and catalogued genes encoding secreted proteins that may contribute to the initial stages of larval establishment, the synthesis of plant growth hormones, extra-oral digestion and evasion of host defenses. A significant portion of the secreted proteins with unknown functions were unique to C. nasturtii and were often members of larger gene families organized in genomic clusters with conservation patterns suggesting that they are undergoing selection.

A Kunitz-type inhibitor from tick salivary glands: A promising novel antitumor drug candidate.

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Transcriptomic analysis of the tick midgut and salivary gland responses upon repeated blood-feeding on a vertebrate host.

Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host’s hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.

Whole-body transcriptome mining for candidate effectors from Diuraphis noxia

BackgroundProteins within aphid saliva play a crucial role as the molecular interface between aphids and their host plants. These salivary effectors modulate plant responses to favour aphid feeding and facilitate infestation. The identification of effectors from economically important pest species is central in understanding the molecular events during the aphid-plant interaction. The Russian wheat aphid (Diuraphis noxia, Kurdjumov) is one such pest that causes devastating losses to wheat and barley yields worldwide. Despite the severe threat to food security posed by D. noxia, the non-model nature of this pest and its host has hindered progress towards understanding this interaction. In this study, in the absence of a salivary gland transcriptome, whole-body transcriptomics data was mined to generate a candidate effector catalogue for D. noxia.ResultsMining the transcriptome identified 725 transcripts encoding putatively secreted proteins amongst which were transcripts specific to D. noxia. Six of the seven examined D. noxia putative effectors, termed DnE’s (Diuraphis noxia effectors) exhibited salivary gland-specific expression. A comparative analysis between whole-body D. noxia transcriptome data versus the head and body transcriptomes from three other aphid species allowed us to define a catalogue of transcripts putatively upregulated in D. noxia head tissue. Five of these were selected for RT-qPCR confirmation, and were found to corroborate the differential expression predictions, with a further three confirmed to be highly expressed in D. noxia salivary gland tissue.ConclusionsDetermining a putative effector catalogue for D. noxia from whole-transcriptome data, particularly the identification of salivary-specific sequences potentially unique to D. noxia, provide the basis for future functional characterisation studies to gain further insight into this aphid-plant interaction. Furthermore, due to a lack of publicly available aphid salivary gland transcriptome data, the capacity to use comparative transcriptomics to compile a list of putative effector candidates from whole-body transcriptomics data will further the study of effectors in various aphid species.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

A reproducible and sensitive method for generating high-quality transcriptomes from single whitefly salivary glands and other low-input tissues.

Transcriptomic studies are an important tool for understanding the molecular pathways underlying host plant use by agricultural pests, including vectors of damaging plant pathogens. Thus far, bulk RNA-Seq has been the main approach for non-model insects. This method relies on pooling large numbers of whole organisms or hundreds of individually dissected organs. The latter approach is logistically challenging, may introduce artifacts of handling and storage, and is not compatible with biological replication. Here, we tested an approach to generate transcriptomes of individual salivary glands and other low-input body tissues from whiteflies (Bemisia tabaci MEAM1), which are major vectors of plant viruses. By comparing our outputs to published bulk RNA-Seq datasets for whole whitefly bodies and pools of salivary glands, we demonstrate that this approach recovers similar numbers of transcripts relative to bulk RNA-Seq in a tissue-specific manner, and for some metrics, exceeds performance of bulk tissue RNA-Seq. Libraries generated from individual salivary glands also yielded additional novel transcripts not identified in pooled salivary gland datasets, and had hundreds of enriched transcripts when compared with whole head tissues. Overall, our study demonstrates that it is feasible to produce high quality, replicated transcriptomes of whitefly salivary glands and other low-input tissues. We anticipate that our approach will expand hypothesis-driven research on salivary glands of whiteflies and other Hemiptera, thus enabling novel control strategies to disrupt feeding and virus transmission.

Screening Candidate Effectors of the Bean Bug Riptortus pedestris by Proteomic and Transcriptomic Analyses

The damage of Riptortus pedestris is exceptional by leading soybean plants to keep green in late autumn. Identification of the salivary proteins is essential to understand how the pest-plant interaction occurs. Here, we have tried to identify them by a combination of proteomic and transcriptomic analyses. The transcriptomes of salivary glands from R. pedestris males, females and nymphs showed about 28,000 unigenes, in which about 40% had open reading frames (ORFs). Therefore, the predicted proteins in the transcriptomes with secretion signals were obtained. Many of the top 1,000 expressed transcripts were involved in protein biosynthesis and transport, suggesting that the salivary glands produce a rich repertoire of proteins. In addition, saliva of R. pedestris males, females and nymphs was collected and proteins inside were identified. In total, 155, 20, and 11 proteins were, respectively, found in their saliva. We have tested the tissue-specific expression of 68 genes that are likely to be effectors, either because they are homologs of reported effectors of other sap-feeding arthropods, or because they are within the top 1,000 expressed genes or found in the salivary proteomes. Their potential functions in regulating plant defenses were discussed. The datasets reported here represent the first step in identifying effectors of R. pedestris.

Comparative analyses of the venom components in the salivary gland transcriptomes and saliva proteomes of some heteropteran insects.

Salivary gland-specific transcriptomes of nine heteropteran insects with distinct feeding strategies (predaceous, hematophagous, and phytophagous) were analyzed and annotated to compare and identify the venom components as well as their expression profiles. The transcriptional abundance of venom genes was verified via quantitative real-time PCR. Hierarchical clustering of 30 representative differentially expressed venom genes from the nine heteropteran species revealed unique groups of salivary gland-specific genes depending on their feeding strategy. The commonly transcribed genes included a paralytic neurotoxin (arginine kinase), digestive enzymes (cathepsin and serine protease), an anti-inflammatory protein (cystatin), hexamerin, and an odorant binding protein. Both predaceous and hematophagous (bed bug) heteropteran species showed relatively higher transcription levels of genes encoding proteins involved in proteolysis and cytolysis, whereas phytophagous heteropterans exhibited little or no expression of these genes, but had a high expression of vitellogenin, a multifunctional allergen. Saliva proteomes from four representative species were also analyzed. All venom proteins identified via saliva proteome analysis were annotated using salivary gland transcriptome data. The proteomic expression profiles of venom proteins were in good agreement with the salivary gland-specific transcriptomic profiles. Our results indicate that profiling of the salivary gland transcriptome provides important information on the composition and evolutionary features of venoms depending on their feeding strategy.

A new dicistro-like virus from soldier fly, Inopus flavus (Diptera: Stratiomyidae), a pest of sugarcane.

Native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the family Dicistroviridae. The complete genome sequence consists of 9793 nucleotides with two open reading frames. The genome includes two potential internal ribosomal entry sites (IRESs): one within the 5' UTR and the other in the intergenic region (IGR). Virus particles purified from infected larvae and visualised by electron microscopy were found to be icosahedral, non-enveloped, and 30 nm in diameter.