A deep insight into the sialome of male and female aedes aegypti mosquitoes

Only adult female mosquitoes feed on blood, while both genders take sugar meals. Accordingly, several compounds associated with blood feeding (i.e. vasodilators, anti-clotting, anti-platelets) are found only in female glands, while enzymes associated with sugar feeding or antimicrobials (such as lysozyme) are found in the glands of both sexes. We performed de novo assembly of reads from adult Aedes aegypti female and male salivary gland libraries (285 and 90 million reads, respectively). By mapping back the reads to the assembled contigs, plus mapping the reads from a publicly available Ae. aegypti library from adult whole bodies, we identified 360 transcripts (including splice variants and alleles) overexpressed tenfold or more in the glands when compared to whole bodies. Moreover, among these, 207 were overexpressed fivefold or more in female vs. male salivary glands, 85 were near equally expressed and 68 were overexpressed in male glands. We call in particular the attention to C-type lectins, angiopoietins, female-specific Antigen 5, the 9.7 kDa, 12–14 kDa, 23.5 kDa, 62/34 kDa, 4.2 kDa, proline-rich peptide, SG8, 8.7 kDa family and SGS fragments: these polypeptides are all of unknown function, but due to their overexpression in female salivary glands and putative secretory nature they are expected to affect host physiology. We have also found many transposons (some of which novel) and several endogenous viral transcripts (probably acquired by horizontal transfer) which are overexpressed in the salivary glands and may play some role in tissue-specific gene regulation or represent a mechanism of virus interference. This work contributes to a near definitive catalog of male and female salivary gland transcripts from Ae. aegypti, which will help to direct further studies aiming at the functional characterization of the many transcripts with unknown function and the understanding of their role in vector-host interaction and pathogen transmission

Anopheline salivary protein genes and gene families: an evolutionary overview after the whole genome sequence of sixteen Anopheles species

Background: Mosquito saliva is a complex cocktail whose pharmacological properties play an essential role in blood feeding by counteracting host physiological response to tissue injury. Moreover, vector borne pathogens are transmitted to vertebrates and exposed to their immune system in the context of mosquito saliva which, in virtue of its immunomodulatory properties, can modify the local environment at the feeding site and eventually affect pathogen transmission. In addition, the host antibody response to salivary proteins may be used to assess human exposure to mosquito vectors. Even though the role of quite a few mosquito salivary proteins has been clarified in the last decade, we still completely ignore the physiological role of many of them as well as the extent of their involvement in the complex interactions taking place between the mosquito vectors, the pathogens they transmit and the vertebrate host. The recent release of the genomes of 16 Anopheles species offered the opportunity to get insights into function and evolution of salivary protein families in anopheline mosquitoes. Results: Orthologues of fifty three Anopheles gambiae salivary proteins were retrieved and annotated from 18 additional anopheline species belonging to the three subgenera Cellia, Anopheles, and Nyssorhynchus. Our analysis included 824 full-length salivary proteins from 24 different families and allowed the identification of 79 novel salivary genes and re-annotation of 379 wrong predictions. The comparative, structural and phylogenetic analyses yielded an unprecedented view of the anopheline salivary repertoires and of their evolution over 100 million years of anopheline radiation shedding light on mechanisms and evolutionary forces that contributed shaping the anopheline sialomes. Conclusions: We provide here a comprehensive description, classification and evolutionary overview of the main anopheline salivary protein families and identify two novel candidate markers of human exposure to malaria vectors worldwide. This anopheline sialome catalogue, which is easily accessible as hyperlinked spreadsheet, is expected to be useful to the vector biology community and to improve the capacity to gain a deeper understanding of mosquito salivary proteins facilitating their possible exploitation for epidemiological and/or pathogen-vector-host interaction studies

Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure

Background : Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. Methodology/Principal Findings : A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). Conclusion/Significance : We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck

Tick and host derived compounds detected in the cement complex substance

Ticks are obligate hematophagous arthropods and vectors of pathogens affecting human and animal health worldwide. Cement is a complex protein polymerization substance secreted by ticks with antimicrobial properties and a possible role in host attachment, sealing the feeding lesion, facilitating feeding and pathogen transmission, and protection from host immune and inflammatory responses. The biochemical properties of tick cement during feeding have not been fully characterized. In this study, we characterized the proteome of Rhipicephalus microplus salivary glands (sialome) and cement (cementome) together with their physicochemical properties at different adult female parasitic stages. The results showed the combination of tick and host derived proteins and other biomolecules such as a-Gal in cement composition, which varied during the feeding process. We propose that these compounds may synergize in cement formation, solidification and maintenance to facilitate attachment, feeding, interference with host immune response and detachment. These results advanced our knowledge of the complex tick cement composition and suggested that tick and host derived compounds modulate cement properties throughout tick feeding

An Insight into the Sialome of the Lone Star Tick, \u3ci\u3eAmbylomma americanum\u3c/i\u3e, With a Glimpse On Its Time Dependent Gene Expression

Hard ticks feed for several days or weeks on their hosts. Blood feeding is assisted by tick saliva, which is injected in the host skin regularly, alternating with blood ingestion. Tick saliva contains hundreds or thousands of different peptides and other bioactive compounds that assist feeding by inhibiting their hosts’ blood clotting, platelet aggregation, vasoconstriction, as well as pain and itching. Immunomodulatory and antimicrobial peptides are also found in tick saliva. Molecular characterization of tick salivary compounds, or its sialome (from the Greek sialos = saliva), helps identification of possible antigens that might confer anti-tick immunity, as well as identifying novel pharmacologically active compounds. Amblyomma americanum is a major nuisance tick in Eastern and Southern US, being a vector of Theileria and Ehrlichia bacteria to animals and humans. Presently we report an RNAseq study concerning the salivary glands of adult female A. americanum ticks, which involved sequencing of four libraries collected at different times of feeding. A total of 5,792 coding sequences were deduced from the transcriptome assembly, 3,139 of which were publicly deposited, expanding from the previously available 146 salivary sequences found in GenBank. A remarkable time-dependent transcript expression was found, mostly related to secretory products, supporting the idea that ticks may have several “sialomes” that are expressed at different times during feeding. The molecular nature of this sialome switching remains unknown. The hyperlinked spreadsheet containing the deduced coding sequences can be found at http://exon.niaid.nih.gov/transcriptome/Amb_americanum/Ambame-web.xlsx

Scenes From Tick Physiology: Proteins of Sialome Talk About Their Biological Processes

Ticks are blood-sucking parasites with different strategies of feeding depending on the tick family. The major families are Ixodidae or Argasidae, being slow or fast feeders, respectively. In the recent years, the advances in molecular sequencing techniques have enabled to gain knowledge about the proteome of the tick''s salivary glands. But an holistic view of the biological processes underlying the expression of the sialome has been neglected. In this study we propose the use of standard biological processes as a tool to draw the physiology of the tick''s salivary glands. We used published data on the sialome of Rhipicephalus sanguineus s.l. (Ixodidae) and Ornithodoros rostratus (Argasidae). A partial set of proteins obtained by these studies were used to define the biological process(es) in which proteins are involved. We used a directed network construction in which the nodes are proteins (source) and biological processes (target), separately for the low-level processes ("children") and the top-level ones ("parents"). We applied the method to feeding R. sanguineus at different time slices, and to different organs of O. rostratus. The network connects the proteins and the processes with a strength directly proportional to the transcript per millions of each protein. We used PageRank as a measure of the importance of each biological process. As suggested in previous studies, the sialome of unfed R. sanguineus express about 30% less biological processes than feeding ticks. Another decrease (25%) is noticed at the middle of the feeding and before detachment. However, top-level processes are deeply affected only at the onset of feeding, demonstrating a redundancy in the feeding. When ixodid-argasid are compared, large differences were observed: they do not share 91% of proteins, but share 90% of the biological processes. However, caution must be observed when examining these results. The hypothesis of different proteins linked to similar biological process(es) in both ticks is an extreme not confirmed in this study. Considering the limitations of this study, carried out with a selected set of proteins, we propose the networks of proteins of sialome linked to their biological processes as a tool aimed to explain the biological processes behind families of proteins

Exploring the transcriptomic data of the Australian paralysis tick, Ixodes holocyclus

Ixodes holocyclus is the paralysis tick commonly found in Australia. I. holocyclus does not cause paralysis in the primary host – bandicoots, but markedly affects secondary hosts such as companion animals, livestock and humans. Holocyclotoxins are the neurotoxin molecules in I. holocyclus responsible for paralysis symptoms. There is a limited understanding of holocyclotoxins due to the difficulties in purifying and expressing these toxins in vitro. Next-generation sequencing technologies were utilised for the first time to generate transcriptome data from two cDNA samples –salivary glands samples collected from female adult ticks engorged on paralysed companion animals and on bandicoots. Contigencoded proteins in each library were annotated according to their best BLAST match against several databases and functionally assigned into six protein categories: housekeeping, transposable elements, pathogen-related, hypothetical, secreted and novel. The “secreted protein” category is comprised of ten protein families: enzymes, protease inhibitors, antigens, mucins, immunity-related, lipocalins, glycinerich, putative secreted, salivary and toxin-like. Comparisons of contig representation between the two libraries reveal the differential expression of tick proteins collected from different hosts. This study provides a preliminary description of the I. holocyclus tick salivary gland transcriptome

Exploring the transcriptomic data of the Australian paralysis tick, Ixodes holocyclus

Ixodes holocyclus is the paralysis tick commonly found in Australia. I. holocyclus does not cause paralysis in the primary host – bandicoots, but markedly affects secondary hosts such as companion animals, livestock and humans. Holocyclotoxins are the neurotoxin molecules in I. holocyclus responsible for paralysis symptoms. There is a limited understanding of holocyclotoxins due to the difficulties in purifying and expressing these toxins in vitro. Next-generation sequencing technologies were utilised for the first time to generate transcriptome data from two cDNA samples –salivary glands samples collected from female adult ticks engorged on paralysed companion animals and on bandicoots. Contigencoded proteins in each library were annotated according to their best BLAST match against several databases and functionally assigned into six protein categories: housekeeping, transposable elements, pathogen-related, hypothetical, secreted and novel. The “secreted protein” category is comprised of ten protein families: enzymes, protease inhibitors, antigens, mucins, immunity-related, lipocalins, glycinerich, putative secreted, salivary and toxin-like. Comparisons of contig representation between the two libraries reveal the differential expression of tick proteins collected from different hosts. This study provides a preliminary description of the I. holocyclus tick salivary gland transcriptome

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes

Introduction: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. Results: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. Conclusion: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.This work was supported by the Intramural Research Program of the Division of Intramural Research (AI001246 and AI000810), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). Open Access funding provided by the National Institutes of Health (NIH).S