Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae) worldwide. The subgenus Adlerius is taxonomically challenging and currently comprises about 20 species with a wide geographic distribution from eastern Asia to southeastern Europe. Some species are confirmed or suspected vectors of Leishmania donovani/infantum, L. major, and L. tropica, and are thus of high medical and veterinary relevance. A single record of Phlebotomus (Adlerius) simici in Austria from 2018 marks its sporadic northernmost and westernmost occurrence, with the origin of its appearance remaining unclear. To better understand Adlerius diversification and particularly post-glacial spread of Ph. simici to northern parts of Europe, we combined phylogenetic analyses with climatic suitability modelling. Divergence time estimates well supported the currently observed geographic distribution of the studied species and revealed several taxonomic challenges in the subgenus. We clearly delineated three distinct genetic and geographic Ph. simici lineages and phylogeographically assessed diversification that were well supported by climatic models. This study provides a comprehensive phylogenetic analysis of the subgenus Adlerius, enhancing our understanding of the diversification in relation to changing climate of this understudied group, and we present new insights into the post-glacial spread of Ph. simici, a suspected vector of L. infantum.

• Keywords
• Adlerius, COI, Central Europe, Divergence time, Phylogeography,
• MeSH
• Phylogeny MeSH
• Phylogeography MeSH
• Insect Vectors * genetics   classification   parasitology   MeSH
• Phlebotomus * genetics   classification   MeSH
• Climate * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH

BACKGROUND: Phlebotomine sand flies are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). Information on sand flies in Central Europe is scarce and, to date, in Austria, only Phlebotomus mascittii has been recorded. In 2018 and 2019, entomological surveys were conducted in Austria with the aim to further clarify sand fly distribution and species composition. RESULTS: In 2019, a Ph. simici specimen was trapped in Austria for the first time. Analyses of two commonly used marker genes, cytochrome c oxidase I (coxI) and cytochrome b (cytb), revealed high sequence identity with Ph. simici specimens from North Macedonia and Greece. Phylogenetic analyses showed high intraspecific distances within Ph. simici, thereby dividing this species into three lineages: one each from Europe, Turkey and Israel. Low interspecific distances between Ph. simici, Ph. brevis and an as yet unidentified Adlerius sp. from Turkey and Armenia highlight how challenging molecular identification within the Adlerius complex can be, even when standard marker genes are applied. CONCLUSION: To our knowledge, this study reports the first finding of Ph. simici in Austria, representing the northernmost recording of this species to date. Moreover, it reveals valuable insights into the phylogenetic relationships among species within the subgenus Adlerius. Phlebotomus simici is a suspected vector of L. infantum and therefore of medical and veterinary importance. Potential sand fly expansion in Central Europe due to climatic change and the increasing import of Leishmania-infected dogs from endemic areas support the need for further studies on sand fly distribution in Austria and Central Europe in general.

• Keywords
• Adlerius, Central europe, Leishmania infantum, Phlebotomine sand fly, Refugial area,
• MeSH
• Cytochromes b genetics   MeSH
• Phylogeny MeSH
• Insect Vectors classification   genetics   MeSH
• Genes, Insect MeSH
• Disease Vectors MeSH
• Classification MeSH
• Leishmaniasis, Visceral transmission   MeSH
• Phlebotomus * classification   genetics   MeSH
• Psychodidae * classification   genetics   MeSH
• Electron Transport Complex IV genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Australia MeSH
• Names of Substances
• Cytochromes b MeSH
• Electron Transport Complex IV MeSH

BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

• Keywords
• DNA barcoding, Ethiopia, MALDI-TOF mass spectrometry, Morphology, Phlebotomus, Protein profiling, Sand flies,
• MeSH
• Species Specificity MeSH
• Phylogeny MeSH
• Insect Vectors classification   MeSH
• Leishmaniasis MeSH
• Psychodidae classification   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Ethiopia epidemiology   MeSH

BACKGROUND: The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. METHODS: All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. RESULTS: Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. CONCLUSIONS: The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

• Keywords
• Crete, Greece, Phlebotominae, Phlebotomus (Adlerius) creticus n. sp., Sand fly fauna,
• MeSH
• Species Specificity MeSH
• Insect Vectors physiology   MeSH
• Leishmaniasis, Cutaneous transmission   MeSH
• Leishmaniasis, Visceral transmission   MeSH
• Phlebotomus anatomy & histology   classification   physiology   MeSH
• Psychodidae anatomy & histology   classification   parasitology   MeSH
• Seasons MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Greece MeSH

Leishmania rely heavily on glycans to complete their digenetic life cycle in both mammalian and phlebotomine sand fly hosts. Leishmania promastigotes secrete a proteophosphoglycan-rich gel (Promastigote Secretory Gel, PSG) that is regurgitated during transmission and can exacerbate infection in the skin. Here we explored the role of PSG from natural Leishmania-sand fly vector combinations by obtaining PSG from Leishmania (L.) major-infected Phlebotomus (P.) papatasi and P. duboscqi and L. tropica-infected P. arabicus. We found that, in addition to the vector's saliva, the PSG from L. major and L. tropica potently exacerbated cutaneous infection in BALB/c mice, improved the probability of developing a patent cutaneous lesion, parasite growth and the evolution of the lesion. Of note, the presence of PSG in the inoculum more than halved the prepatent period of cutaneous L. tropica infection from an average of 32 weeks to 13 weeks. In addition, L. major and L. tropica PSG extracted from the permissive experimental vector, Lutzomyia (Lu.) longipalpis, also exacerbated infections in mice. These results reinforce and extend the hypothesis that PSG is an important and evolutionarily conserved component of Leishmania infection that can be used to facilitate experimental infection for drug and vaccine screening.

• Keywords
• Cutaneous leishmaniasis, Leishmania, Leishmania major, Leishmania mexicana, Leishmania tropica, PSG, sand fly, transmission, zoonoses,
• MeSH
• Skin drug effects   parasitology   pathology   MeSH
• Leishmania major chemistry   MeSH
• Leishmania tropica chemistry   MeSH
• Leishmaniasis, Cutaneous parasitology   pathology   MeSH
• Membrane Proteins administration & dosage   chemistry   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Parasite Load MeSH
• Phlebotomus parasitology   MeSH
• Proteoglycans administration & dosage   chemistry   MeSH
• Protozoan Proteins administration & dosage   chemistry   MeSH
• Saliva MeSH
• Symptom Flare Up MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Ppg1 protein, Leishmania MeSH Browser
• Proteoglycans MeSH
• Protozoan Proteins MeSH

Leishmania spp. are medically important unicellular parasites transmitted by phlebotomine sand flies. The World Health Organization recently highlighted the importance of reliable diagnostic tools for leishmaniasis. Our study of human infection was conducted in two endemic foci of Leishmania tropica in the Galilee region, northern Israel. Elevated anti-Leishmania antibodies were present in the majority (78.6%) of L. tropica-PCR positive individuals. Moreover, the enzyme-linked immunosorbent assay showed high sensitivity, specificity, and negative and positive predictive values (ranging between 73% and 79%), thus fulfilling the basic requirement for future development of a serodiagnostic and screening tool. The anti-sand fly saliva antibodies used as biomarkers of exposure reflected the composition of the local sand fly fauna as well as the abundance of individual species. High levels of antibodies against vector salivary proteins may further indicate frequent exposure to sand flies and consequently a higher probability of Leishmania transmission.

• MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Leishmania tropica * MeSH
• Leishmaniasis, Cutaneous diagnosis   epidemiology   MeSH
• Humans MeSH
• Psychodidae parasitology   MeSH
• Sensitivity and Specificity MeSH
• Seroepidemiologic Studies MeSH
• Serologic Tests methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Israel epidemiology   MeSH

BACKGROUND: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. RESULTS: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. CONCLUSIONS: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

• Keywords
• Leishmania amazonensis, Lipophosphoglycan, Lutzomyia longipalpis, Lutzomyia migonei, Phlebotomus papatasi, Vector-parasite interaction,
• MeSH
• Glycosphingolipids analysis   MeSH
• Leishmania chemistry   growth & development   isolation & purification   MeSH
• Humans MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycosphingolipids MeSH
• lipophosphonoglycan MeSH Browser

BACKGROUND: Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. RESULTS: We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. CONCLUSIONS: We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut.

• Keywords
• Glycoprotein, Leishmania, Lipophosphoglycan, Phlebotomine sand flies,
• MeSH
• Glycoconjugates genetics   metabolism   MeSH
• Insect Vectors metabolism   parasitology   MeSH
• Insect Proteins genetics   metabolism   MeSH
• Leishmania physiology   MeSH
• Mucins genetics   metabolism   MeSH
• Psychodidae genetics   metabolism   parasitology   MeSH
• Digestive System metabolism   parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycoconjugates MeSH
• Insect Proteins MeSH
• Mucins MeSH

BACKGROUND: The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. METHODOLOGY AND PRINCIPAL FINDINGS: Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? CONCLUSIONS AND SIGNIFICANCE: We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

• MeSH
• Biological Evolution * MeSH
• History, 19th Century MeSH
• History, 20th Century MeSH
• History, 21st Century MeSH
• History, Ancient MeSH
• Insect Vectors parasitology   MeSH
• Leishmania classification   isolation & purification   MeSH
• Leishmaniasis epidemiology   history   MeSH
• Psychodidae classification   growth & development   MeSH
• Disease Reservoirs parasitology   MeSH
• Fossils MeSH
• Animals MeSH
• Check Tag
• History, 19th Century MeSH
• History, 20th Century MeSH
• History, 21st Century MeSH
• History, Ancient MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Review MeSH

Phlebotomus sergenti Parrot, 1917 is the main vector of Leishmania tropica; however, its broad geographical range and molecular heterogeneity suggest possible variability in vector competence. We infected laboratory-reared P. sergenti originating from Turkey and Israel to compare their susceptibility to L. tropica. In both tested groups, heavy late-stage infections with the presence of metacyclic forms and colonization of the stomodeal valve were observed. The similar development of Leishmania in both sand fly colonies indicates that the different geographical origin of P. sergenti is not reflected by a different vector competence to L. tropica. Additionally, we tested the effect of the gregarine Psychodiella sergenti on L. tropica coinfections; no apparent differences were found between P. sergenti infected or not infected by gregarines.

• Keywords
• Leishmania tropica, Phlebotomus sergenti, coinfection, gregarine, vector competence,
• MeSH
• Apicomplexa physiology   MeSH
• Insect Vectors parasitology   MeSH
• Host-Parasite Interactions * MeSH
• Leishmania tropica growth & development   MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH