Transcriptome changes in humans acutely exposed to nanoparticles during grinding of dental nanocomposites.

Aim: Today, there is a lack of research studies concerning human acute exposure to nanoparticles (NPs). Our investigation aimed to simulate real-world acute inhalation exposure to NPs released during work with dental nanocomposites in a dental office or technician laboratory. Methods: Blood samples from female volunteers were processed before and after inhalation exposure. Transcriptomic mRNA and miRNA expression changes were analyzed. Results: We detected large interindividual variability, 90 significantly deregulated mRNAs, and 4 miRNAs when samples of participants before and after dental nanocomposite grinding were compared. Conclusion: The results suggest that inhaled dental NPs may present an occupational hazard to human health, as indicated by the changes in the processes related to oxidative stress, synthesis of eicosanoids, and cell division.

What is this article about? We searched for a possible impact of acute inhalation exposure to nanoparticles (NPs) released during the grinding of dental nanocomposites used for teeth reconstruction. The exposure design utilized in our study simulated the acute exposure of the dental staff to the NPs. Our research fills the gaps in knowledge in the field of acute human inhalation exposure to dental nanocomposites.What were the results? Results indicate that the impact of exposure to NPs is dependent on the style of working as well as on the interindividual biological variability among study subjects. Changes in expression levels of genes associated with an increase of oxidative stress, synthesis of eicosanoids (signaling molecules related to e.g., immune responses), and cell division were detected.What do the results of the study mean? All the observed changes may contribute to the pathogenesis of neurodegenerative disorders, carcinogenesis, or problems during pregnancy. Occupational exposure to inhaled NPs, including those generated in dental practice can pose a significant health risk, and protective measures when working with these materials should be considered. More research is needed to compare our results with chronic (long-term) exposure to similar materials to show the hazards related to their inhalation.

Gene expression profiles and protein-protein interaction networks in THP-1 cells exposed to metal-based nanomaterials.

We analyzed gene expression in THP-1 cells exposed to metal-based nanomaterials (NMs) [TiO2 (NM-100), ZnO (NM-110), SiO2 (NM-200), Ag (NM-300 K)]. A functional enrichment analysis of the significant differentially expressed genes (DEGs) identified the key modulated biological processes and pathways. DEGs were used to construct protein-protein interaction networks. NM-110 and NM-300 K induced changes in the expression of genes involved in oxidative and genotoxic stress, immune response, alterations of cell cycle, detoxification of metal ions and regulation of redox-sensitive pathways. Both NMs shared a number of highly connected protein nodes (hubs) including CXCL8, ATF3, HMOX1, and IL1B. NM-200 induced limited transcriptional changes, mostly related to the immune response; however, several hubs (CXCL8, ATF3) were identical with NM-110 and NM-300 K. No effects of NM-100 were observed. Overall, soluble nanomaterials NM-110 and NM-300 K exerted a wide variety of toxic effects, while insoluble NM-200 induced immunotoxicity; NM-100 caused no detectable changes on the gene expression level.

Sympatry in a nightingale contact zone has no effect on host-specific blood parasite prevalence and lineage diversity.

Parasites are a key driving force behind many ecological and evolutionary processes. Prevalence and diversity of parasites, as well as their effects on hosts, are not uniform across host species. As such, the potential parasite spillover between species can significantly influence outcomes of interspecific interactions. We screened two species of Luscinia nightingales for haemosporidian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) along an approximately 3000 km transect in Europe, incorporating areas of host distant allopatry, close allopatry and sympatry. We found significant differences in infection rates between the two host species, with common nightingales having much lower parasite prevalence than thrush nightingales (36.7% versus 83.8%). This disparity was mostly driven by Haemoproteus prevalence, which was significantly higher in thrush nightingales while common nightingales had a small, but significantly higher, Plasmodium prevalence. Furthermore, we found no effect of proximity to the contact zone on infection rate in either host species. Despite having lower infection prevalence, common nightingales were infected with a significantly higher diversity of parasite lineages than thrush nightingales, and lineage assemblages differed considerably between the two species, even in sympatry. This pattern was mostly driven by the large diversity of comparatively rare lineages, while the most abundant lineages were shared between the two host species. This suggests that, despite the close evolutionary relationships between the two nightingales, there are significant differences in parasite prevalence and diversity, regardless of the distance from the contact zone. This suggests that spillover of haemosporidian blood parasites is unlikely to contribute towards interspecific interactions in this system.

Transcriptomic alterations in the olfactory bulb induced by exposure to air pollution: Identification of potential biomarkers and insights into olfactory system function.

This study evaluated how exposure to the ubiquitous air pollution component, ultrafine particles (UFPs), alters the olfactory bulb (OB) transcriptome. The study utilised a whole-body inhalation chamber to simulate real-life conditions and focused on UFPs due to their high translocation and deposition ability in OBs as well as their prevalence in ambient air. Female C57BL/6J mice were exposed to clean air or to freshly generated combustion derived UFPs for two weeks, after which OBs were dissected and mRNA transcripts were investigated using RNA sequencing analysis. For the first time, transcriptomics was applied to determine changes in mRNA expression levels occurring after subacute exposure to UFPs in the OBs. We found forty-five newly described mRNAs to be involved in air pollution-induced responses, including genes involved in odorant binding, synaptic regulation, and myelination signalling pathway, providing new gene candidates for future research. This study provides new insights for the environmental science and neuroscience fields and nominates future research directions.

Metal Nanoparticles with Antimicrobial Properties: The Toxicity Response in Mouse Mesenchymal Stem Cells.

Some metal nanoparticles (NP) are characterized by antimicrobial properties with the potential to be used as alternative antibiotics. However, NP may negatively impact human organism, including mesenchymal stem cells (MSC), a cell population contributing to tissue growth and regeneration. To address these issues, we investigated the toxic effects of selected NP (Ag, ZnO, and CuO) in mouse MSC. MSC were treated with various doses of NP for 4 h, 24 h, and 48 h and multiple endpoints were analyzed. Reactive oxygen species were generated after 48 h CuO NP exposure. Lipid peroxidation was induced after 4 h and 24 h treatment, regardless of NP and/or tested dose. DNA fragmentation and oxidation induced by Ag NP showed dose responses for all the periods. For other NP, the effects were observed for shorter exposure times. The impact on the frequency of micronuclei was weak. All the tested NP increased the sensitivity of MSC to apoptosis. The cell cycle was most affected after 24 h, particularly for Ag NP treatment. In summary, the tested NP induced numerous adverse changes in MSC. These results should be taken into consideration when planning the use of NP in medical applications where MSC are involved.

Effects of various environments on epigenetic settings and chromosomal damage.

Air pollution is a dominant environmental exposure factor with significant health consequences. Unexpectedly, research in a heavily polluted region of the Czech Republic, with traditional heavy industry, revealed repeatedly the lowest frequency of micronuclei in the season with the highest concentrations of air pollutants including carcinogenic benzo[a]pyrene (B[a]P). Molecular findings have been collected for more than 10 years from various locations of the Czech Republic, with differing quality of ambient air. Preliminary conclusions have suggested adaptation of the population from the polluted locality (Ostrava, Moravian-Silesian Region (MSR)) to chronic air pollution exposure. In this study we utilize the previous findings and, for the first time, investigate micronuclei (MN) frequency by type: (i) centromere positive (CEN+) MN, representing chromosomal losses, and (ii) centromere negative (CEN-) MN representing chromosomal breaks. As previous results indicated differences between populations in the expression of XRCC5, a gene involved in the non-homologous end-joining (NHEJ) repair pathway, possible variations in epigenetic settings in this gene were also investigated. This new research was conducted in two seasons in the groups from two localities with different air quality levels (Ostrava (OS) and Prague (PG)). The obtained new results show significantly lower frequencies of chromosomal breaks in the OS subjects, related to the highest air pollution levels (p < 0.001). In contrast, chromosomal losses were comparable between both groups. In addition, significantly lower DNA methylation was found in 14.3% of the analyzed CpG loci of XRCC5 in the population from OS. In conclusion, the epigenetic adaptation (hypomethylation) in XRCC5 involved in the NHEJ repair pathway in the population from the polluted region, was suggested as a reason for the reduced level of chromosomal breaks. Further research is needed to explore the additional mechanisms, including genetic adaptation.

The impact of extractable organic matter from gasoline and alternative fuel emissions on bronchial cell models (BEAS-2B, MucilAir™).

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAir™). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

Markers of lipid oxidation and inflammation in bronchial cells exposed to complete gasoline emissions and their organic extracts.

Road traffic emissions consist of gaseous components, particles of various sizes, and chemical compounds that are bound to them. Exposure to vehicle emissions is implicated in the etiology of inflammatory respiratory disorders. We investigated the inflammation-related markers in human bronchial epithelial cells (BEAS-2B) and a 3D model of the human airways (MucilAir™), after exposure to complete emissions and extractable organic matter (EOM) from particles generated by ordinary gasoline (E5), and a gasoline-ethanol blend (E20; ethanol content 20% v/v). The production of 22 lipid oxidation products (derivatives of linoleic and arachidonic acid, AA) and 45 inflammatory molecules (cytokines, chemokines, growth factors) was assessed after days 1 and 5 of exposure, using LC-MS/MS and a multiplex immunoassay, respectively. The response observed in MucilAir™ exposed to E5 gasoline emissions, characterized by elevated levels of pro-inflammatory AA metabolites (prostaglandins) and inflammatory markers, was the most pronounced. E20 EOM exposure was associated with increased levels of AA metabolites with anti-inflammatory effects in this cell model. The exposure of BEAS-2B cells to complete emissions reduced lipid oxidation, while E20 EOM tended to increase concentrations of AA metabolite and chemokine production; the impacts on other inflammatory markers were limited. In summary, complete E5 emission exposure of MucilAir™ induces the processes associated with the pro-inflammatory response. This observation highlights the potential negative health impacts of ordinary gasoline, while the effects of alternative fuel are relatively weak.

The Impact of Air Pollution Exposure on the MicroRNA Machinery and Lung Cancer Development.

Small non-coding RNA molecules (miRNAs) play an important role in the epigenetic regulation of gene expression. As these molecules have been repeatedly implicated in human cancers, they have been suggested as biomarkers of the disease. Additionally, miRNA levels have been shown to be affected by environmental pollutants, including airborne contaminants. In this review, we searched the current literature for miRNAs involved in lung cancer, as well as miRNAs deregulated as a result of exposure to air pollutants. We then performed a synthesis of the data and identified those molecules commonly deregulated under both conditions. We detected a total of 25 miRNAs meeting the criteria, among them, miR-222, miR-21, miR-126-3p, miR-155 and miR-425 being the most prominent. We propose these miRNAs as biomarkers of choice for the identification of human populations exposed to air pollution with a significant risk of developing lung cancer.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.

Amine-binding properties of salivary yellow-related proteins in phlebotomine sand flies.

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phlebotomus perniciosus and P. orientalis, the Old-World vectors of visceral leishmaniases causative agents. Expressed and purified YRPs from three different sand fly species were tested for their interactions with various biogenic amines, including serotonin, histamine and catecholamines. Using the L. longipalpis YRP LJM11 as a control, we have demonstrated the comparability of the microscale thermophoresis method with conventional isothermal titration calorimetry described previously. By homology in silico modeling, we predicted the surface charge and both amino acids and hydrogen bonds of the amine-binding motifs to influence the binding affinities between closely related YRPs. All YRPs tested bound at least two biogenic amines, while the affinities differ both among and within species. Low affinity was observed for histamine. The salivary recombinant proteins rSP03B (P. perniciosus) and rPorASP4 (P. orientalis) showed high-affinity binding of serotonin, suggesting their capability to facilitate inhibition of the blood vessel contraction and platelet aggregation.

Synthetic peptides as a novel approach for detecting antibodies against sand fly saliva.

BACKGROUND: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. METHODOLOGY/PRINCIPAL FINDINGS: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. CONCLUSIONS/SIGNIFICANCE: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations.

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Insights into the sand fly saliva: Blood-feeding and immune interactions between sand flies, hosts, and Leishmania.

BACKGROUND: Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. METHODS AND FINDINGS: In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. CONCLUSIONS: In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host-sand fly-parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae).

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

BACKGROUND: Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. CONCLUSIONS/SIGNIFICANCE: Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Comparative analysis of salivary gland transcriptomes of Phlebotomus orientalis sand flies from endemic and non-endemic foci of visceral leishmaniasis.

BACKGROUND: In East Africa, Phlebotomus orientalis serves as the main vector of Leishmania donovani, the causative agent of visceral leishmaniasis (VL). Phlebotomus orientalis is present at two distant localities in Ethiopia; Addis Zemen where VL is endemic and Melka Werer where transmission of VL does not occur. To find out whether the difference in epidemiology of VL is due to distant compositions of P. orientalis saliva we established colonies from Addis Zemen and Melka Werer, analyzed and compared the transcriptomes, proteomes and enzymatic activity of the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Two cDNA libraries were constructed from the female salivary glands of P. orientalis from Addis Zemen and Melka Werer. Clones of each P. orientalis library were randomly selected, sequenced and analyzed. In P. orientalis transcriptomes, we identified members of 13 main protein families. Phylogenetic analysis and multiple sequence alignments were performed to evaluate differences between the P. orientalis colonies and to show the relationship with other sand fly species from the subgenus Larroussius. To further compare both colonies, we investigated the humoral antigenicity and cross-reactivity of the salivary proteins and the activity of salivary apyrase and hyaluronidase. CONCLUSIONS: This is the first report of the salivary components of P. orientalis, an important vector sand fly. Our study expanded the knowledge of salivary gland compounds of sand fly species in the subgenus Larroussius. Based on the phylogenetic analysis, we showed that P. orientalis is closely related to Phlebotomus tobbi and Phlebotomus perniciosus, whereas Phlebotomus ariasi is evolutionarily more distinct species. We also demonstrated that there is no significant difference between the transcriptomes, proteomes or enzymatic properties of the salivary components of Addis Zemen (endemic area) and Melka Werer (non-endemic area) P. orientalis colonies. Thus, the different epidemiology of VL in these Ethiopian foci cannot be attributed to the salivary gland composition.