BACKGROUND
phlebotomine sand flies are the arthropod vectors of leishmania parasites, the causative agents of leishmaniasis.
during the feeding process sand flies inject saliva into the site of the bite to facilitate successful acquisition of a blood meal  <cit> .
an infected sand fly regurgitates infective metacyclic promastigote stage leishmania while feeding; thus, the parasite is always introduced to the host as a mixture with sand fly saliva.
sand fly saliva facilitates the transmission of leishmania parasites to mammalian hosts; at the same time, immune response to salivary components was shown to partially protect the host from leishmania infection  <cit> .
therefore, salivary components essential for parasite transmission and/or eliciting protective immune response are sought-after.
salivary proteins from phlebotomus papatasi, the vector of leishmania major, and lutzomyia longipalpis, the vector of l. infantum, have been extensively studied.
in addition, cdna libraries from several other sand fly species were characterized and include other sand flies that vector l. major , l. infantum  and l. donovani .
phlebotomus  arabicus is distributed in certain parts of east africa and the middle east.
in ethiopia, p. arabicus females infected with uncharacterized leishmania sp.
were reported  <cit> , and in northern israel p. arabicus is the proven vector of cutaneous leishmaniasis caused by l. tropica  <cit> .
cutaneous leishmaniasis caused by l. tropica is found in a vast discontinuous area reaching from the south-western mediterranean to turkey, north-western india and sub-saharan africa  <cit> .
long supposed to circulate in anthroponotic foci exclusively, l. tropica was recently shown to occur as an anthropozoonosis as well  <cit> .
laboratory experiments demonstrated that p. arabicus is a permissive vector, meaning it is susceptible to development of more than one species of leishmania, including l. major and l. infantum  <cit> .
in the present study, salivary gland transcripts and proteins of p.  arabicus were studied by cdna sequencing, electrophoretic and proteomic methods.
this is the first study of the repertoire of salivary molecules of a vector of l. tropica and it is the first report of the composition of salivary proteins in the subgenus adlerius.
RESULTS
sequencing of salivary gland cdna library
a cdna library was constructed from salivary glands of phlebotomus arabicus females dissected one day after emergence.
from this cdna library,  <dig> random transcripts were selected and sequenced, resulting in  <dig> high quality sequences.
sequences were clustered together based on sequence homology and produced  <dig> clusters and  <dig> sequences were assessed as singletons .
the term cluster may refer to either singletons or multiple homologous sequences.
similar to other sand flies studied so far, the most abundant transcripts were those coding for putative secretory proteins and resulted in  <dig> clusters with an average number of  <dig>  sequences per cluster.
predicted proteins containing retention signals for endoplasmic reticulum and/or transmembrane domains were not treated as putatively secreted.
an example of such proteins is the translocon-associated protein complex, δ subunit , which has homologs previously designated as  <dig> kda or  <dig>  kda salivary protein in p. ariasi or l. longipalpis, respectively.
members of  <dig> different families were found among putative secretory proteins.
blast comparison of translated nucleotide sequences with the nr protein database showed that overall, high similarity was observed namely with salivary proteins of l. infantum vectors p.  ariasi and p.  perniciosus.
the expected values of these matches were highly significant at values lower than e- <dig>  to a lesser extent, similarity to sequences of salivary proteins of p.  argentipes, the vector of l. donovani in india, was also observed.
these findings are fully in concert with the close evolutionary relationship of larroussius and adlerius subgenera reported by aransay et al.
<cit> .
some of the protein families contained multiple members.
the observed variability among individual protein family members might be explained by intraspecific polymorphism, as the tested sample was heterogeneous .
nevertheless, analysis of genetic variation of sp <dig> salivary protein in p. papatasi brought strong evidence that sp- <dig> is a multicopy gene  <cit> .
while individual intraspecific variability of sand fly salivary proteins awaits broader analysis, we propose that the multiple homologous transcripts within protein families observed in this p. arabicus salivary gland cdna library may reflect gene duplication events or allelic variation.
full-length sequences were obtained for most clusters coding for putatively secreted proteins.
only sequences containing a signal peptide and a polya tail in the coding cdna were considered full-length.
table  <dig> lists clusters for which full length sequences were obtained, including the name of the sequence, the best match to ncbi nr database, the predicted molecular weight  and isoelectric point  of the mature protein, and the genbank accession number of the nucleotide coding sequence.
the table also includes information on the presence of individual proteins in p. arabicus salivary proteome, as confirmed by edman degradation and/or mass spectrometry.
a more detailed description of the putative secreted proteins follows, starting with proteins encoded by the most abundant transcripts:
detection of a protein in the proteome that matches the predicted peptide sequence of the transcript is denoted under protein detected by "y"
sp15-like proteins
thus far, sp15-like proteins have only been reported in sand flies and their function remains unknown.
it was suggested that sp15-like proteins were derived from an ancestral odorant-binding protein and were closely related to short d <dig> proteins  <cit> .
immunization of mice with p. papatasi sp <dig> conferred partial protection against l. major infection  <cit> .
transcripts coding for these proteins represented the most abundant family in p. arabicus salivary gland cdna library and clustered into three separate groups .
the amino acid sequences of mature proteins coded by p. arabicus transcripts share  <dig> % amino acid identity and  <dig> % amino acid similarity.
when sp15-like proteins from other sand flies were added to the analysis, only the six cysteines and three other amino acids were conserved in the amino acid sequence of mature proteins , reflecting the previously reported divergence among sp15-like proteins in sand flies  <cit> .
in l. longipalpis a single sp15-like protein was found, sl <dig>  in p. arabicus and other phlebotomus spp.
studied so far multiple members of the sp <dig> family are present.
a phylogenetic analysis revealed three separate groups of p. arabicus sp15-like proteins, showing close relationships to p. ariasi proteins parsp <dig>  parsp <dig> and par <dig>  respectively .
the predicted pi of all three p. arabicus sp15-like variants is highly basic , corresponding to the fact that most phlebotomus spp.
sand fly salivary proteins have very high predicted pi values.
<dig> kda and  <dig> kda proteins
six clusters coding for proteins related to p. ariasi  <dig> kda salivary protein  and p. perniciosus  <dig> kda salivary protein  were found in the p. arabicus salivary gland cdna library.
there are no other homologs of these proteins in accessible databases, no conserved domains were found in the translated sequences, and no function has been assigned to these proteins.
however, in p. arabicus cdna library they represent the second most abundant protein family.
transcripts coding for parsp25-like proteins occurred in long  and short forms , with very little variability among individual clusters.
the mature proteins coded by these transcripts have a predicted mw of  <dig> kda and  <dig> kda, respectively, and are composed of  <dig> % identical amino acids and  <dig> % similar amino acids.
the predicted pi of the proteins is acidic , unlike most sand fly salivary proteins described thus far.
d7-related proteins
d <dig> proteins are well known from the saliva of mosquitoes, sand flies, black flies and biting midges  <cit> .
while the structure of anopheline d <dig> proteins allows binding of biogenic amines and components of the contact activation system of coagulation  <cit> , related proteins in sand flies lack conserved residues responsible for stabilizing bound ligands  <cit> .
thus, they may not interfere with host hemostasis by a similar mechanism and their function remains unknown.
mosquito d <dig> proteins elicit ige in individuals hypersensitive to mosquito bites  <cit>  and antibodies against sand fly d <dig> proteins were found in dogs naturally exposed to l. longipalpis  <cit> .
thus, it is possible that sand fly d <dig> proteins are involved in human hypersensitivity to sand fly bites.
four clusters of sequences coding for d7-related proteins were found in the p. arabicus salivary gland cdna library .
predicted mature proteins have mw of 26– <dig> kda and an average basic pi of  <dig> .
two of the seven clusters have potential n-glycosylation sites, as predicted by netnglyc server.
the protein sequences of mature proteins were  <dig> % identical and  <dig> % similar .
the phylogenetic analysis showed four distinct clades among p. arabicus d7-related proteins, all of them bearing high similarity to p. ariasi and p. perniciosus proteins .
yellow-related proteins
yellow-related proteins are common in insects; in bloodsucking diptera, yellow-related proteins were described from mosquitoes and sand flies.
in ae.
aegypti, a dopachrome-converting enzyme shares homology with drosophila melanogaster yellow proteins  <cit>  and, according to li et al.
<cit> , it might play a role in melanotic encapsulation of parasites in the hemocoel.
in sand fly salivary gland samples, however, dopachrome-converting enzyme activity could not be detected , while yellow protein of p. duboscqi was detected in midgut and salivary glands and shown to have lectin properties  <cit> .
sand fly yellow proteins were previously proposed as potential antigens recognized by sera of experimentally bitten mice and dogs, and naturally exposed humans  <cit> .
in l. longipalpis this was also suggested by mass spectrometry  <cit> .
in the p. arabicus salivary gland cdna library a single homolog of yellow-related proteins was found .
the predicted mw of the protein is  <dig>  kda with a pi of  <dig> .
no n-glycosylation sites were predicted in the protein sequence by amino acid submission to the netnglyc server.
ppsp32-like proteins
ppsp32-like proteins, so named due to homology with proteins described from p. papatasi, have only been found in sand flies and their function is unknown.
in p. perniciosus they possess a collagen-related internal sequence  <cit> .
in p. arabicus, however, these proteins bear no significant similarity to collagen; this feature is shared with ppsp32-like proteins of p. papatasi, p. ariasi or p. argentipes.
similarly to other protein families analyzed, the phylogenetic position of p. arabicus ppsp32-like proteins is close to that of p. ariasi and p. perniciosus homologs .
three different transcript clusters coding for ppsp32-like proteins were found in the p. arabicus salivary gland cdna library , the mature proteins being  <dig> % identical .
the variable length of the central glycine-rich region of the protein sequence results in three different variants of mature proteins.
the predicted mw of the three variants are  <dig>   <dig>  and  <dig>  kda.
in all three variants of these proteins, there are alternating regions of very acidic  and very basic  amino acids .
as shown in figure 3c, the basic regions include the central glycine-rich sequence and the c-terminal basic tail.
no n-glycosylation sites were predicted for these proteins by the netnglyc server.
antigen 5-related proteins
antigen  <dig>  protein is present in vespid venom  <cit>  and related proteins were reported in the saliva of bloodsucking insects  <cit> .
similar to most other sand fly species studied so far, only one cluster coding for ag5-related protein was found in the p. arabicus cdna library   <cit> .
mature ag5-related proteins of sand flies are  <dig> % identical and  <dig> % similar, overall .
the phylogenetic analysis of ag5-related proteins from sand flies, other blood-feeding insects and selected hymenopteran species shows a strongly supported distinct clade of sand fly ag5-related proteins .
unlike previous reports  <cit> , this sand fly clade does not contain any culicoides sequences.
close relationship of p. arabicus ag5-related protein to p. perniciosus and p. ariasi was observed, much in the same way as in other salivary protein families .
the predicted mw of the mature protein is  <dig>  kda and the pi is very basic .
apyrase
apyrases are widespread in saliva of bloodsucking insects.
the antihemostatic effects of saliva are, for a great part, due to apyrase anti-platelet activity  <cit> .
sand fly apyrases belong to the cimex apyrase family  <cit> .
three very similar apyrase clusters coding for apyrases were found in p. arabicus cdna library  .
the predicted average pi of p. arabicus apyrases is  <dig>  and the predicted mw is  <dig>  kda.
endonuclease
a cluster encoding a putative endonuclease was identified in the p. arabicus cdna library .
similar sequences were reported from p. ariasi, p. perniciosus, p. argentipes, and l. longipalpis salivary glands.
cluster pabsp <dig> encodes an endonuclease domain, which is typical for dna/rna non-specific endonucleases.
since all residues composing the active site, the substrate binding site and the mg2+ binding site are conserved in this cluster; we suggest that pabsp <dig> might possess endonuclease activity.
the predicted pi of the mature protein is  <dig>  and the predicted mw of the mature protein is  <dig>  kda.
possible roles for a salivary endonuclease include reducing the viscosity of the blood pool during feeding and liberating nucleosides.
exogenous nucleosides, primarily adenosine, can exhibit regulatory effects on blood clotting, immune and inflammatory responses, and leishmania pathogenesis  <cit> .
hyaluronidase
hyaluronidase activity has been detected in several species of bloodsucking insects including sand flies  <cit> .
accessible expressed sequence tag  data from cdna libraries of p. papatasi and p. duboscqi salivary glands do not contain hyaluronidase transcripts.
nonetheless, the enzyme activity was detected in salivary gland samples from these species  <cit> , highlighting the potent enzymatic activity of a protein produced from a low abundance transcript.
in salivary gland homogenate of p. arabicus, hyaluronidase activity was also observed.
as revealed by zymography, the apparent molecular weight of the p. arabicus hyaluronidase holoenzyme is approximately  <dig> kda , but no protein band correspond to the predicted molecular weight could be detected by silver or coomassie staining in electrophoretically separated salivary proteins.
these observations reflect the scarcity of both hyaluronidase transcript and hyaluronidase protein in sand fly salivary glands, and at the same time underline the remarkably high specific activity of the enzyme.
in p. arabicus, the predicted pi for mature hyaluronidase is  <dig>  and the mw is  <dig> kda.
additional putative enzymes
in the amino acid translation of sequence cluster  <dig> , a phospholipase a <dig>  domain is present, containing all conserved residues of both catalytic and metal-binding sites of pla <dig>  in hymenopteran venoms, pla <dig> represents a major allergen.
in the salivary glands of blood-feeding insects, sequences coding for pla2-like proteins were reported only from sand flies of the subgenus larroussius  <cit> .
we tested salivary gland samples of p. arabicus specifically for pla <dig> activity and did not detect any positive reaction.
cluster  <dig> contains an exceptionally long 5' untranslated region  compared to other clusters from this cdna library coding for secreted proteins.
the 5' utr in this cluster is more than  <dig> nucleotides long.
thus, the regulation of expression of this transcript might be different from other transcripts reported herein.
other sequences coding for other putative enzymes could not be obtained as full-length clones.
these included a pyrophosphatase-like protein , amylase , an enzyme involved in digestion of dietary starch  <cit> , and trehalase .
previously, sequences coding for pyrophosphatase-like proteins were reported in p. argentipes and p. duboscqi sand flies  <cit> .
these proteins, as well as their p. arabicus homolog reported herein, contain a conserved phosphodiesterase domain, typical for enzymes cleaving phosphodiester and phosphosulphate bonds in nad, deoxynucleotides and nucleotide sugars  <cit> .
transcripts coding for α-amylase were found in l. longipalpis salivary glands and midguts as well as p. papatasi midguts  <cit> .
amylase activity was shown in l. longipalpis and p. papatasi salivary gland samples  <cit>  and it is likely that the enzymatic activity is present also in p. arabicus salivary glands.
the putative trehalase enzyme from p. arabicus salivary glands might either be an intrinsic component of insect metabolism, or might be related to sugar feeding and digestion.
trehalose is the main energy source in insect hemolymph in general.
trehalases are involved in its hydrolysis, yielding glucose molecules which are then readily available to various cells of the insect body  <cit> .
so far, trehalase enzyme or sequence has not been reported from salivary glands of any blood-feeding insects, but sequences coding for sand fly trehalase have been found in midgut cdna libraries of p. papatasi  <cit> .
putative secreted proteins of unknown function
there were a number of transcripts with no homology to known enzymes or structural proteins; however, eight of these transcripts encode potentially secreted proteins with high homology to other sand fly salivary molecules.
p. arabicus salivary transcripts code for  <dig> kda proteins homologous to parsp <dig>  polymorphisms resulting in different translations of the transcripts were observed .
these proteins are seemingly sand fly-specific; apart from  <dig> sand fly species ; no other related proteins from any organism were reported.
another family of putative sand fly-specific proteins contain homologs of p. arabicus  <dig> kda salivary protein .
transcripts coding for such proteins were reported from p. perniciosus, p. ariasi, p. duboscqi and l. longipalpis.
homologs of p. arabicus  <dig>  kda peptide  were only found in p. ariasi and p. perniciosus  <cit> .
our finding contradicts the suggestion that  <dig>  kda peptides are specific for the subgenus larroussius  <cit> .
likewise, two unrelated  <dig> kda proteins  were found in p. arabicus; homologous molecules were identified in p. ariasi  <cit> .
additionally, two transcripts putatively encoding  <dig> kda salivary proteins  are homologous to molecules identified in p. argentipes  <cit> .
two sequence clusters coding for putatively secreted proteins in the p. arabicus cdna library show no similarity with known sand fly sequences.
cluster  <dig>  is homologous to ae.
aegypti putative salivary secreted mucin  <dig>  as well as the ige binding protein icarapin from honeybee venom  <cit> .
the predicted molecular weight of the mature protein is  <dig>  kda and the putative protein would have an acidic pi of  <dig> .
there are  <dig> potential n-glycosylation sites and  <dig> potential o-glycosylation sites in the amino acid sequence of cluster  <dig>  as predicted by submission to the netnglyc and netoglyc servers.
similarly, putative extracellular proteins of anopheles gambiae  and aedes aegypti  were also predicted to contain multiple o-glycosylation sites.
these proteins might be involved in hypersensitivity to bites of blood-feeding insects.
the second cluster  <dig> , encodes a homolog of conserved hypothetical proteins of culicine as well as anopheline mosquitoes.
the predicted molecular weight of the mature protein from p. arabicus is  <dig>  kda and the predicted pi is  <dig> .
no n- or o-glycosylation sites were predicted in cluster  <dig> protein and nothing is known about these hypothetical proteins.
proteome analysis of p. arabicus salivary glands
for the proteome analysis, p. arabicus salivary gland samples separated by sds-page were subjected to edman degradation and mass spectrometry.
edman degradation resulted in the identification of  <dig> different n-terminal sequences.
these were representative of two  <dig> kda proteins , yellow-related protein , and endonuclease .
an n-terminal sequence common to all six variants of salivary proteins similar to parsp <dig> was also detected by edman degradation , as well as n-terminal sequences common to apyrases  and to d7-related proteins a and c .
from the data obtained by edman degradation analysis it could not be concluded which variants of polymorphic salivary proteins were present in the proteome.
mass spectrometry was used for a more detailed analysis of p. arabicus salivary proteome.
by this method,  <dig> putative secreted proteins were identified in the proteome .
these proteins include amylase , yellow-related protein , two  <dig> kda salivary proteins , all three apyrase-like proteins , two ppsp32-like proteins , antigen 5-related protein , four  <dig> kda salivary proteins similar to parsp <dig> , three d7-related proteins , and two ppsp15-like proteins .
in addition, one high-molecular weight protein  analyzed by mass spectrometry revealed no similarity to predicted p. arabicus secreted salivary proteins.
we assume this protein represents a component of salivary gland wall rather than a secreted protein present in the saliva.
accordingly, in p. duboscqi female salivary glands, we previously detected multiple protein bands running at molecular weight protein > <dig> kda which were specifically present in the wall of salivary glands  <cit> .
additionally, glycoprotein-specific staining of electrophoretically separated proteins was performed.
proq emerald staining detected six glycoprotein bands in p. arabicus salivary gland samples .
three bands  correlate with proteins identified by mass spectrometry: amylase , yellow-related protein , and  <dig> kda proteins .
band a is predicted to migrate at about  <dig> kda and may represent hyaluronidase; however, this band may be produced by the oligomerization of other salivary proteins or components of the gland structure.
bands e and f do not distinctly correlate with molecules identified by mass spectrometry and are therefore unknown.
humoral response to p. arabicus saliva
some of the proteins homologous to p. arabicus salivary proteins are known as antigens or allergens in other insect species.
p. arabicus salivary proteins elicit a strong antibody response in mice exposed to p. arabicus feeding.
in western blots, the most prominent antigenic bands recognized by sera of two bitten mice  had apparent molecular weights of 56– <dig>  kda,  <dig> kda,  <dig> kda ,  <dig> kda,  <dig> – <dig>  kda and  <dig> kda.
slightly weaker reactions were observed with bands running at  <dig> kda and  <dig>  kda.
in addition, sera from some animals recognized two very faint bands, running at  <dig> kda and  <dig> kda .
CONCLUSIONS
in this study we generated a transcriptome of female sand fly phlebotomus arabicus salivary glands using a pcr-based cdna library.
this is the first reported salivary gland transcriptome of a sand fly from the subgenus adlerius.
the most abundant transcripts were represented in the  <dig> high quality sequences.
many of the transcripts encoded full- or partial-length proteins; most of which are homologous to other sand fly species saliva molecules.
phylogenetic analysis consistently shows a strong relationship between p. arabicus with sand flies from the larroussius subgenus; specifically, p. ariasi and p. perniciousus.
the phylogenetic analyses of sand fly salivary proteins reaffirm the taxonomy of phlebotomines  <cit> .
some of the most abundant molecules identified in the transcriptome that have a predicted signal secretion peptide include a  <dig> kda protein , a d7-related protein , a yellow-related protein , an antigen 5-related protein  and  <dig> kda and  <dig> kda proteins similar to p. ariasi parsp <dig> .
a number of paralogous transcripts were identified, such as those in the sp <dig> and d <dig> families.
the presence of duplicate gene copies has been observed in other blood feeding arthropods  <cit>  and can serve several potential functions including increased transcript abundance and rapid evolution of blood feeding strategies while retaining intrinsic proteins.
proteomic analysis by n-terminal sequencing or tryptic digestion followed by mass spectrometry identified  <dig> proteins in the salivary gland homogenate of p. arabicus that were characterized in the transcriptome.
in addition, one protein was identified by mass spectrometry that did not match any of the characterized transcripts.
this is the most comprehensive description of sand fly salivary proteome to date and also demonstrates that the transcriptome represents >95% of the most abundant proteins present in the salivary gland.
in the analysis of the p. arabicus salivary gland transcriptome four sequences were identified as encoding a putative hyaluronidase.
hyaluronidase is an enzyme that has been identified in a number of phlebotomine salivary glands including lutzomyia longipalpis, p. phlebotomus papatasi, p. phlebotomus duboscqi, p. paraphlebotomus sergenti and p. adlerius halepensis  <cit> .
the zymographic analysis of salivary gland extract confirms the presence of an active hyaluronidase enzyme and demonstrates the effectiveness of a transcriptomic approach to identifying disease vector salivary components.
having described the repertoire of saliva molecules opens more doors in the research of vector-host and vector-parasite interactions, pharmacology and insect biochemistry.
the antigenicity of sand fly saliva is one important aspect of the vector-host interaction.
evaluating the humoral response of mice bitten by p. arabicus demonstrates the abundance and diversity of antigenic molecules in the saliva.
future work may focus on the use of functional transcriptomics  to use these p. arabicus salivary proteins to evaluate the role of these molecules in the epidemiology of leishmaniasis.

 phlebotomine sand flies are the arthropod vectors of leishmania parasites, the causative agents of leishmaniasis. during the feeding process sand flies inject saliva into the site of the bite to facilitate successful acquisition of a blood meal. an infected sand fly regurgitates infective metacyclic promastigote stage leishmania while feeding; thus, the parasite is always introduced to the host as a mixture with sand fly saliva. sand fly saliva facilitates the transmission of leishmania parasites to mammalian hosts; at the same time, immune response to salivary components was shown to partially protect the host from leishmania infection. therefore, salivary components essential for parasite transmission and/or eliciting protective immune response are sought-after. salivary proteins from phlebotomus papatasi, the vector of leishmania major, and lutzomyia longipalpis, the vector of l. infantum, have been extensively studied. in addition, cdna libraries from several other sand fly species were characterized and include other sand flies that vector l. major, l. infantum and l. donovani. phlebotomus arabicus is distributed in certain parts of east africa and the middle east. in ethiopia, p. arabicus females infected with uncharacterized leishmania sp. were reported, and in northern israel p. arabicus is the proven vector of cutaneous leishmaniasis caused by l. tropica. cutaneous leishmaniasis caused by l. tropica is found in a vast discontinuous area reaching from the south-western mediterranean to turkey, north-western india and sub-saharan africa. long supposed to circulate in anthroponotic foci exclusively, l. tropica was recently shown to occur as an anthropozoonosis as well. laboratory experiments demonstrated that p. arabicus is a permissive vector, meaning it is susceptible to development of more than one species of leishmania, including l. major and l. infantum. in the present study, salivary gland transcripts and proteins of p. arabicus were studied by cdna sequencing, electrophoretic and proteomic methods. this is the first study of the repertoire of salivary molecules of a vector of l. tropica and it is the first report of the composition of salivary proteins in the subgenus adlerius.  sequencing of salivary gland cdna library a cdna library was constructed from salivary glands of phlebotomus arabicus females dissected one day after emergence. from this cdna library, random transcripts were selected and sequenced, resulting in high quality sequences. sequences were clustered together based on sequence homology and produced clusters and sequences were assessed as singletons. the term cluster may refer to either singletons or multiple homologous sequences. similar to other sand flies studied so far, the most abundant transcripts were those coding for putative secretory proteins and resulted in clusters with an average number of sequences per cluster. predicted proteins containing retention signals for endoplasmic reticulum and/or transmembrane domains were not treated as putatively secreted. an example of such proteins is the translocon-associated protein complex, δ subunit, which has homologs previously designated as kda or kda salivary protein in p. ariasi or l. longipalpis, respectively. members of different families were found among putative secretory proteins. blast comparison of translated nucleotide sequences with the nr protein database showed that overall, high similarity was observed namely with salivary proteins of l. infantum vectors p. ariasi and p. perniciosus. the expected values of these matches were highly significant at values lower than e- to a lesser extent, similarity to sequences of salivary proteins of p. argentipes, the vector of l. donovani in india, was also observed. these findings are fully in concert with the close evolutionary relationship of larroussius and adlerius subgenera reported by aransay et al.. some of the protein families contained multiple members. the observed variability among individual protein family members might be explained by intraspecific polymorphism, as the tested sample was heterogeneous. nevertheless, analysis of genetic variation of sp salivary protein in p. papatasi brought strong evidence that sp- is a multicopy gene. while individual intraspecific variability of sand fly salivary proteins awaits broader analysis, we propose that the multiple homologous transcripts within protein families observed in this p. arabicus salivary gland cdna library may reflect gene duplication events or allelic variation. full-length sequences were obtained for most clusters coding for putatively secreted proteins. only sequences containing a signal peptide and a polya tail in the coding cdna were considered full-length. table lists clusters for which full length sequences were obtained, including the name of the sequence, the best match to ncbi nr database, the predicted molecular weight and isoelectric point of the mature protein, and the genbank accession number of the nucleotide coding sequence. the table also includes information on the presence of individual proteins in p. arabicus salivary proteome, as confirmed by edman degradation and/or mass spectrometry. a more detailed description of the putative secreted proteins follows, starting with proteins encoded by the most abundant transcripts: detection of a protein in the proteome that matches the predicted peptide sequence of the transcript is denoted under protein detected by "y" sp15-like proteins thus far, sp15-like proteins have only been reported in sand flies and their function remains unknown. it was suggested that sp15-like proteins were derived from an ancestral odorant-binding protein and were closely related to short d proteins. immunization of mice with p. papatasi sp conferred partial protection against l. major infection. transcripts coding for these proteins represented the most abundant family in p. arabicus salivary gland cdna library and clustered into three separate groups. the amino acid sequences of mature proteins coded by p. arabicus transcripts share % amino acid identity and % amino acid similarity. when sp15-like proteins from other sand flies were added to the analysis, only the six cysteines and three other amino acids were conserved in the amino acid sequence of mature proteins, reflecting the previously reported divergence among sp15-like proteins in sand flies. in l. longipalpis a single sp15-like protein was found, sl in p. arabicus and other phlebotomus spp. studied so far multiple members of the sp family are present. a phylogenetic analysis revealed three separate groups of p. arabicus sp15-like proteins, showing close relationships to p. ariasi proteins parsp parsp and par respectively. the predicted pi of all three p. arabicus sp15-like variants is highly basic, corresponding to the fact that most phlebotomus spp. sand fly salivary proteins have very high predicted pi values. kda and kda proteins six clusters coding for proteins related to p. ariasi kda salivary protein and p. perniciosus kda salivary protein were found in the p. arabicus salivary gland cdna library. there are no other homologs of these proteins in accessible databases, no conserved domains were found in the translated sequences, and no function has been assigned to these proteins. however, in p. arabicus cdna library they represent the second most abundant protein family. transcripts coding for parsp25-like proteins occurred in long and short forms, with very little variability among individual clusters. the mature proteins coded by these transcripts have a predicted mw of kda and kda, respectively, and are composed of % identical amino acids and % similar amino acids. the predicted pi of the proteins is acidic, unlike most sand fly salivary proteins described thus far. d7-related proteins d proteins are well known from the saliva of mosquitoes, sand flies, black flies and biting midges. while the structure of anopheline d proteins allows binding of biogenic amines and components of the contact activation system of coagulation, related proteins in sand flies lack conserved residues responsible for stabilizing bound ligands. thus, they may not interfere with host hemostasis by a similar mechanism and their function remains unknown. mosquito d proteins elicit ige in individuals hypersensitive to mosquito bites and antibodies against sand fly d proteins were found in dogs naturally exposed to l. longipalpis. thus, it is possible that sand fly d proteins are involved in human hypersensitivity to sand fly bites. four clusters of sequences coding for d7-related proteins were found in the p. arabicus salivary gland cdna library. predicted mature proteins have mw of 26– kda and an average basic pi of. two of the seven clusters have potential n-glycosylation sites, as predicted by netnglyc server. the protein sequences of mature proteins were % identical and % similar. the phylogenetic analysis showed four distinct clades among p. arabicus d7-related proteins, all of them bearing high similarity to p. ariasi and p. perniciosus proteins. yellow-related proteins yellow-related proteins are common in insects; in bloodsucking diptera, yellow-related proteins were described from mosquitoes and sand flies. in ae. aegypti, a dopachrome-converting enzyme shares homology with drosophila melanogaster yellow proteins and, according to li et al., it might play a role in melanotic encapsulation of parasites in the hemocoel. in sand fly salivary gland samples, however, dopachrome-converting enzyme activity could not be detected, while yellow protein of p. duboscqi was detected in midgut and salivary glands and shown to have lectin properties. sand fly yellow proteins were previously proposed as potential antigens recognized by sera of experimentally bitten mice and dogs, and naturally exposed humans. in l. longipalpis this was also suggested by mass spectrometry. in the p. arabicus salivary gland cdna library a single homolog of yellow-related proteins was found. the predicted mw of the protein is kda with a pi of. no n-glycosylation sites were predicted in the protein sequence by amino acid submission to the netnglyc server. ppsp32-like proteins ppsp32-like proteins, so named due to homology with proteins described from p. papatasi, have only been found in sand flies and their function is unknown. in p. perniciosus they possess a collagen-related internal sequence. in p. arabicus, however, these proteins bear no significant similarity to collagen; this feature is shared with ppsp32-like proteins of p. papatasi, p. ariasi or p. argentipes. similarly to other protein families analyzed, the phylogenetic position of p. arabicus ppsp32-like proteins is close to that of p. ariasi and p. perniciosus homologs. three different transcript clusters coding for ppsp32-like proteins were found in the p. arabicus salivary gland cdna library, the mature proteins being % identical. the variable length of the central glycine-rich region of the protein sequence results in three different variants of mature proteins. the predicted mw of the three variants are and kda. in all three variants of these proteins, there are alternating regions of very acidic and very basic amino acids. as shown in figure 3c, the basic regions include the central glycine-rich sequence and the c-terminal basic tail. no n-glycosylation sites were predicted for these proteins by the netnglyc server. antigen 5-related proteins antigen protein is present in vespid venom and related proteins were reported in the saliva of bloodsucking insects. similar to most other sand fly species studied so far, only one cluster coding for ag5-related protein was found in the p. arabicus cdna library. mature ag5-related proteins of sand flies are % identical and % similar, overall. the phylogenetic analysis of ag5-related proteins from sand flies, other blood-feeding insects and selected hymenopteran species shows a strongly supported distinct clade of sand fly ag5-related proteins. unlike previous reports, this sand fly clade does not contain any culicoides sequences. close relationship of p. arabicus ag5-related protein to p. perniciosus and p. ariasi was observed, much in the same way as in other salivary protein families. the predicted mw of the mature protein is kda and the pi is very basic. apyrase apyrases are widespread in saliva of bloodsucking insects. the antihemostatic effects of saliva are, for a great part, due to apyrase anti-platelet activity. sand fly apyrases belong to the cimex apyrase family. three very similar apyrase clusters coding for apyrases were found in p. arabicus cdna library. the predicted average pi of p. arabicus apyrases is and the predicted mw is kda. endonuclease a cluster encoding a putative endonuclease was identified in the p. arabicus cdna library. similar sequences were reported from p. ariasi, p. perniciosus, p. argentipes, and l. longipalpis salivary glands. cluster pabsp encodes an endonuclease domain, which is typical for dna/rna non-specific endonucleases. since all residues composing the active site, the substrate binding site and the mg2+ binding site are conserved in this cluster; we suggest that pabsp might possess endonuclease activity. the predicted pi of the mature protein is and the predicted mw of the mature protein is kda. possible roles for a salivary endonuclease include reducing the viscosity of the blood pool during feeding and liberating nucleosides. exogenous nucleosides, primarily adenosine, can exhibit regulatory effects on blood clotting, immune and inflammatory responses, and leishmania pathogenesis. hyaluronidase hyaluronidase activity has been detected in several species of bloodsucking insects including sand flies. accessible expressed sequence tag data from cdna libraries of p. papatasi and p. duboscqi salivary glands do not contain hyaluronidase transcripts. nonetheless, the enzyme activity was detected in salivary gland samples from these species, highlighting the potent enzymatic activity of a protein produced from a low abundance transcript. in salivary gland homogenate of p. arabicus, hyaluronidase activity was also observed. as revealed by zymography, the apparent molecular weight of the p. arabicus hyaluronidase holoenzyme is approximately kda, but no protein band correspond to the predicted molecular weight could be detected by silver or coomassie staining in electrophoretically separated salivary proteins. these observations reflect the scarcity of both hyaluronidase transcript and hyaluronidase protein in sand fly salivary glands, and at the same time underline the remarkably high specific activity of the enzyme. in p. arabicus, the predicted pi for mature hyaluronidase is and the mw is kda. additional putative enzymes in the amino acid translation of sequence cluster, a phospholipase a domain is present, containing all conserved residues of both catalytic and metal-binding sites of pla in hymenopteran venoms, pla represents a major allergen. in the salivary glands of blood-feeding insects, sequences coding for pla2-like proteins were reported only from sand flies of the subgenus larroussius. we tested salivary gland samples of p. arabicus specifically for pla activity and did not detect any positive reaction. cluster contains an exceptionally long 5' untranslated region compared to other clusters from this cdna library coding for secreted proteins. the 5' utr in this cluster is more than nucleotides long. thus, the regulation of expression of this transcript might be different from other transcripts reported herein. other sequences coding for other putative enzymes could not be obtained as full-length clones. these included a pyrophosphatase-like protein, amylase, an enzyme involved in digestion of dietary starch, and trehalase. previously, sequences coding for pyrophosphatase-like proteins were reported in p. argentipes and p. duboscqi sand flies. these proteins, as well as their p. arabicus homolog reported herein, contain a conserved phosphodiesterase domain, typical for enzymes cleaving phosphodiester and phosphosulphate bonds in nad, deoxynucleotides and nucleotide sugars. transcripts coding for α-amylase were found in l. longipalpis salivary glands and midguts as well as p. papatasi midguts. amylase activity was shown in l. longipalpis and p. papatasi salivary gland samples and it is likely that the enzymatic activity is present also in p. arabicus salivary glands. the putative trehalase enzyme from p. arabicus salivary glands might either be an intrinsic component of insect metabolism, or might be related to sugar feeding and digestion. trehalose is the main energy source in insect hemolymph in general. trehalases are involved in its hydrolysis, yielding glucose molecules which are then readily available to various cells of the insect body. so far, trehalase enzyme or sequence has not been reported from salivary glands of any blood-feeding insects, but sequences coding for sand fly trehalase have been found in midgut cdna libraries of p. papatasi. putative secreted proteins of unknown function there were a number of transcripts with no homology to known enzymes or structural proteins; however, eight of these transcripts encode potentially secreted proteins with high homology to other sand fly salivary molecules. p. arabicus salivary transcripts code for kda proteins homologous to parsp polymorphisms resulting in different translations of the transcripts were observed. these proteins are seemingly sand fly-specific; apart from sand fly species; no other related proteins from any organism were reported. another family of putative sand fly-specific proteins contain homologs of p. arabicus kda salivary protein. transcripts coding for such proteins were reported from p. perniciosus, p. ariasi, p. duboscqi and l. longipalpis. homologs of p. arabicus kda peptide were only found in p. ariasi and p. perniciosus. our finding contradicts the suggestion that kda peptides are specific for the subgenus larroussius. likewise, two unrelated kda proteins were found in p. arabicus; homologous molecules were identified in p. ariasi. additionally, two transcripts putatively encoding kda salivary proteins are homologous to molecules identified in p. argentipes. two sequence clusters coding for putatively secreted proteins in the p. arabicus cdna library show no similarity with known sand fly sequences. cluster is homologous to ae. aegypti putative salivary secreted mucin as well as the ige binding protein icarapin from honeybee venom. the predicted molecular weight of the mature protein is kda and the putative protein would have an acidic pi of. there are potential n-glycosylation sites and potential o-glycosylation sites in the amino acid sequence of cluster as predicted by submission to the netnglyc and netoglyc servers. similarly, putative extracellular proteins of anopheles gambiae and aedes aegypti were also predicted to contain multiple o-glycosylation sites. these proteins might be involved in hypersensitivity to bites of blood-feeding insects. the second cluster, encodes a homolog of conserved hypothetical proteins of culicine as well as anopheline mosquitoes. the predicted molecular weight of the mature protein from p. arabicus is kda and the predicted pi is. no n- or o-glycosylation sites were predicted in cluster protein and nothing is known about these hypothetical proteins. proteome analysis of p. arabicus salivary glands for the proteome analysis, p. arabicus salivary gland samples separated by sds-page were subjected to edman degradation and mass spectrometry. edman degradation resulted in the identification of different n-terminal sequences. these were representative of two kda proteins, yellow-related protein, and endonuclease. an n-terminal sequence common to all six variants of salivary proteins similar to parsp was also detected by edman degradation, as well as n-terminal sequences common to apyrases and to d7-related proteins a and c. from the data obtained by edman degradation analysis it could not be concluded which variants of polymorphic salivary proteins were present in the proteome. mass spectrometry was used for a more detailed analysis of p. arabicus salivary proteome. by this method, putative secreted proteins were identified in the proteome. these proteins include amylase, yellow-related protein, two kda salivary proteins, all three apyrase-like proteins, two ppsp32-like proteins, antigen 5-related protein, four kda salivary proteins similar to parsp, three d7-related proteins, and two ppsp15-like proteins. in addition, one high-molecular weight protein analyzed by mass spectrometry revealed no similarity to predicted p. arabicus secreted salivary proteins. we assume this protein represents a component of salivary gland wall rather than a secreted protein present in the saliva. accordingly, in p. duboscqi female salivary glands, we previously detected multiple protein bands running at molecular weight protein > kda which were specifically present in the wall of salivary glands. additionally, glycoprotein-specific staining of electrophoretically separated proteins was performed. proq emerald staining detected six glycoprotein bands in p. arabicus salivary gland samples. three bands correlate with proteins identified by mass spectrometry: amylase, yellow-related protein, and kda proteins. band a is predicted to migrate at about kda and may represent hyaluronidase; however, this band may be produced by the oligomerization of other salivary proteins or components of the gland structure. bands e and f do not distinctly correlate with molecules identified by mass spectrometry and are therefore unknown. humoral response to p. arabicus saliva some of the proteins homologous to p. arabicus salivary proteins are known as antigens or allergens in other insect species. p. arabicus salivary proteins elicit a strong antibody response in mice exposed to p. arabicus feeding. in western blots, the most prominent antigenic bands recognized by sera of two bitten mice had apparent molecular weights of 56– kda, kda, kda, kda, – kda and kda. slightly weaker reactions were observed with bands running at kda and kda. in addition, sera from some animals recognized two very faint bands, running at kda and kda.  in this study we generated a transcriptome of female sand fly phlebotomus arabicus salivary glands using a pcr-based cdna library. this is the first reported salivary gland transcriptome of a sand fly from the subgenus adlerius. the most abundant transcripts were represented in the high quality sequences. many of the transcripts encoded full- or partial-length proteins; most of which are homologous to other sand fly species saliva molecules. phylogenetic analysis consistently shows a strong relationship between p. arabicus with sand flies from the larroussius subgenus; specifically, p. ariasi and p. perniciousus. the phylogenetic analyses of sand fly salivary proteins reaffirm the taxonomy of phlebotomines. some of the most abundant molecules identified in the transcriptome that have a predicted signal secretion peptide include a kda protein, a d7-related protein, a yellow-related protein, an antigen 5-related protein and kda and kda proteins similar to p. ariasi parsp. a number of paralogous transcripts were identified, such as those in the sp and d families. the presence of duplicate gene copies has been observed in other blood feeding arthropods and can serve several potential functions including increased transcript abundance and rapid evolution of blood feeding strategies while retaining intrinsic proteins. proteomic analysis by n-terminal sequencing or tryptic digestion followed by mass spectrometry identified proteins in the salivary gland homogenate of p. arabicus that were characterized in the transcriptome. in addition, one protein was identified by mass spectrometry that did not match any of the characterized transcripts. this is the most comprehensive description of sand fly salivary proteome to date and also demonstrates that the transcriptome represents >95% of the most abundant proteins present in the salivary gland. in the analysis of the p. arabicus salivary gland transcriptome four sequences were identified as encoding a putative hyaluronidase. hyaluronidase is an enzyme that has been identified in a number of phlebotomine salivary glands including lutzomyia longipalpis, p. phlebotomus papatasi, p. phlebotomus duboscqi, p. paraphlebotomus sergenti and p. adlerius halepensis. the zymographic analysis of salivary gland extract confirms the presence of an active hyaluronidase enzyme and demonstrates the effectiveness of a transcriptomic approach to identifying disease vector salivary components. having described the repertoire of saliva molecules opens more doors in the research of vector-host and vector-parasite interactions, pharmacology and insect biochemistry. the antigenicity of sand fly saliva is one important aspect of the vector-host interaction. evaluating the humoral response of mice bitten by p. arabicus demonstrates the abundance and diversity of antigenic molecules in the saliva. future work may focus on the use of functional transcriptomics to use these p. arabicus salivary proteins to evaluate the role of these molecules in the epidemiology of leishmaniasis. 
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 kda-like proteins. enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis. a cdna library from salivary glands of p. arabicus females was constructed and transcripts were sequenced and analyzed. 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. in the present work, we focused on phlebotomus arabicus, which was recently shown to transmit leishmania tropica, the causative agent of cutaneous leishmaniasis in israel. 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. humoral response of mice bitten by p. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic. sand fly saliva plays an important role in blood feeding and leishmania transmission as it was shown to increase parasite virulence. 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.
 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 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 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 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. 