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1.  The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies 
BMC Genomics  2011;12:223.
Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles.
A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal.
This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.
PMCID: PMC3107814  PMID: 21569254
2.  Transcriptomic and functional analysis of the Anopheles gambiae salivary gland in relation to blood feeding 
BMC Genomics  2010;11:566.
The Anopheles gambiae salivary glands play a major role in malaria transmission and express a variety of bioactive components that facilitate blood-feeding by preventing platelet aggregation, blood clotting, vasodilatation, and inflammatory and other reactions at the probing site on the vertebrate host.
We have performed a global transcriptome analysis of the A. gambiae salivary gland response to blood-feeding, to identify candidate genes that are involved in hematophagy. A total of 4,978 genes were found to be transcribed in this tissue. A comparison of salivary gland transcriptomes prior to and after blood-feeding identified 52 and 41 transcripts that were significantly up-regulated and down-regulated, respectively. Ten genes were further selected to assess their role in the blood-feeding process using RNAi-mediated gene silencing methodology. Depletion of the salivary gland genes encoding D7L2, anophelin, peroxidase, the SG2 precursor, and a 5'nucleotidase gene significantly increased probing time of A. gambiae mosquitoes and thereby their capacity to blood-feed.
The salivary gland transcriptome comprises approximately 38% of the total mosquito transcriptome and a small proportion of it is dynamically changing already at two hours in response to blood feeding. A better understanding of the salivary gland transcriptome and its function can contribute to the development of pathogen transmission control strategies and the identification of medically relevant bioactive compounds.
PMCID: PMC3091715  PMID: 20946652
3.  An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus 
BMC Genomics  2010;11:450.
Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R.sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences.
Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained ~56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as "unknown function". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library.
Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.
PMCID: PMC3091647  PMID: 20650005
4.  The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense 
BMC Genomics  2010;11:363.
Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks.
cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology.
We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.
PMCID: PMC2901319  PMID: 20529354
5.  Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus 
BMC Genomics  2009;10:282.
Sand fly saliva plays an important role in blood feeding and Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.
A cDNA library from salivary glands of P. arabicus females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic.
This transcriptomic analysis of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.
PMCID: PMC2714351  PMID: 19555500
6.  Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae) 
BMC Genomics  2008;9:552.
Ticks are obligate blood feeders. The midgut is the first major region of the body where blood and microbes ingested with the blood meal come in contact with the tick's internal tissues. Little is known about protein expression in the digestive tract of ticks. In this study, for analysis of global gene expression during tick attachment and feeding, we generated and sequenced 1,679 random transcripts (ESTs) from cDNA libraries from the midguts of female ticks at varying stages of feeding.
Sequence analysis of the 1,679 ESTs resulted in the identification of 835 distinct transcripts, from these, a total of 82 transcripts were identified as proteins putatively directly involved in blood meal digestion, including enzymes involved in oxidative stress reduction/antimicrobial activity/detoxification, peptidase inhibitors, protein digestion (cysteine-, aspartic-, serine-, and metallo-peptidases), cell, protein and lipid binding including mucins and iron/heme metabolism and transport. A lectin-like protein with a high match to lectins in other tick species, allergen-like proteins and surface antigens important in pathogen recognition and/or antimicrobial activity were also found. Furthermore, midguts collected from the 6-day-fed ticks expressed twice as many transcripts involved in bloodmeal processing as midguts from unfed/2-day-fed ticks.
This tissue-specific transcriptome analysis provides an opportunity to examine the global expression of transcripts in the tick midgut and to compare the gut response to host attachment versus blood feeding and digestion. In contrast to those in salivary glands of other Ixodid ticks, most proteins in the D. variabilis midgut cDNA library were intracellular. Of the total ESTs associated with a function, an unusually large number of transcripts were associated with peptidases, cell, lipid and protein binding, and oxidative stress or detoxification. Presumably, this is consistent with their role in intracellular processing of the blood meal and response to microbial infections. The presence of many proteins with similar functions is consistent with the hypothesis that gene duplication contributed to the successful adaptation of ticks to hematophagy. Furthermore, these transcripts may be useful to scientists investigating the role of the tick midgut in blood-meal digestion, antimicrobial activity or the transmission of tick-borne pathogens.
PMCID: PMC2644717  PMID: 19021911
7.  Insight into the sialome of the castor bean tick, Ixodes ricinus 
BMC Genomics  2008;9:233.
In recent years, there have been several sialome projects revealing transcripts expressed in the salivary glands of ticks, which are important vectors of several human diseases. Here, we focused on the sialome of the European vector of Lyme disease, Ixodes ricinus.
In the attempt to describe expressed genes and their dynamics throughout the feeding period, we constructed cDNA libraries from four different feeding stages of Ixodes ricinus females: unfed, 24 hours after attachment, four (partially fed) and seven days (fully engorged) after attachment. Approximately 600 randomly selected clones from each cDNA library were sequenced and analyzed. From a total 2304 sequenced clones, 1881 sequences forming 1274 clusters underwent subsequent functional analysis using customized bioinformatics software. Clusters were sorted according to their predicted function and quantitative comparison among the four libraries was made. We found several groups of over-expressed genes associated with feeding that posses a secretion signal and may be involved in tick attachment, feeding or evading the host immune system. Many transcripts clustered into families of related genes with stage-specific expression. Comparison to Ixodes scapularis and I. pacificus transcripts was made.
In addition to a large number of homologues of the known transcripts, we obtained several novel predicted protein sequences. Our work contributes to the growing list of proteins associated with tick feeding and sheds more light on the dynamics of the gene expression during tick feeding. Additionally, our results corroborate previous evidence of gene duplication in the evolution of ticks.
PMCID: PMC2410133  PMID: 18489795
8.  The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies 
BMC Genomics  2008;9:15.
In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi.
Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein.
This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.
PMCID: PMC2249575  PMID: 18194529
9.  Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania major-infected sandflies 
BMC Genomics  2007;8:300.
In sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR.
Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major.
This tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by real-time PCR, suggesting that this customized bioinformatic analysis is a powerful and accurate tool useful in analysing expression profiles from different cDNA libraries. Additionally, the findings presented in this work suggest that the Leishmania parasite is modulating key enzymes or proteins in the gut of the sandfly that may be beneficial for its establishment and survival.
PMCID: PMC2034597  PMID: 17760985
10.  cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome 
BMC Genomics  2007;8:255.
The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.
We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.
Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.
PMCID: PMC1978503  PMID: 17662120
11.  High degree of conservancy among secreted salivary gland proteins from two geographically distant Phlebotomus duboscqi sandflies populations (Mali and Kenya) 
BMC Genomics  2006;7:226.
Salivary proteins from sandflies are potential targets for exploitation as vaccines to control Leishmania infection; in this work we tested the hypothesis that salivary proteins from geographically distant Phlebotomus duboscqi sandfly populations are highly divergent due to the pressure exerted by the host immune response. Salivary gland cDNA libraries were prepared from wild-caught P. duboscqi from Mali and recently colonised flies of the same species from Kenya.
Transcriptome and proteome analysis resulted in the identification of the most abundant salivary gland-secreted proteins. Orthologues of these salivary proteins were identified by phylogenetic tree analysis. Moreover, comparative analysis between the orthologues of these two different populations resulted in a high level of protein identity, including the predicted MHC class II T-cell epitopes from all these salivary proteins.
These data refute the hypothesis that salivary proteins from geographically distinct populations of the same Phlebotomus sandfly species are highly divergent. They also suggest the potential for using the same species-specific components in a potential vector saliva-based vaccine.
PMCID: PMC1574310  PMID: 16952314
12.  Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis 
BMC Genomics  2006;7:52.
Immune responses to sandfly saliva have been shown to protect animals against Leishmania infection. Yet very little is known about the molecular characteristics of salivary proteins from different sandflies, particularly from vectors transmitting visceral leishmaniasis, the fatal form of the disease. Further knowledge of the repertoire of these salivary proteins will give us insights into the molecular evolution of these proteins and will help us select relevant antigens for the development of a vector based anti-Leishmania vaccine.
Two salivary gland cDNA libraries from female sandflies Phlebotomus argentipes and P. perniciosus were constructed, sequenced and proteomic analysis of the salivary proteins was performed. The majority of the sequenced transcripts from the two cDNA libraries coded for secreted proteins. In this analysis we identified transcripts coding for protein families not previously described in sandflies. A comparative sandfly salivary transcriptome analysis was performed by using these two cDNA libraries and two other sandfly salivary gland cDNA libraries from P. ariasi and Lutzomyia longipalpis, also vectors of visceral leishmaniasis. Full-length secreted proteins from each sandfly library were compared using a stand-alone version of BLAST, creating formatted protein databases of each sandfly library. Related groups of proteins from each sandfly species were combined into defined families of proteins. With this comparison, we identified families of salivary proteins common among all of the sandflies studied, proteins to be genus specific and proteins that appear to be species specific. The common proteins included apyrase, yellow-related protein, antigen-5, PpSP15 and PpSP32-related protein, a 33-kDa protein, D7-related protein, a 39- and a 16.1- kDa protein and an endonuclease-like protein. Some of these families contained multiple members, including PPSP15-like, yellow proteins and D7-related proteins suggesting gene expansion in these proteins.
This comprehensive analysis allows us the identification of genus- specific proteins, species-specific proteins and, more importantly, proteins common among these different sandflies. These results give us insights into the repertoire of salivary proteins that are potential candidates for a vector-based vaccine.
PMCID: PMC1434747  PMID: 16539713

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