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Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.

Background Phlebotomine sand flies (Diptera: Psychodidae) are haematophagous insects that transmit the protozoan parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), the main causative agent of both zoonotic visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean basin. Eight species of sand flies have been previously recorded in Romania: Phlebotomus papatasi , Phlebotomus alexandri , Phlebotomus sergenti , Phlebotomus perfiliewi , Phlebotomus neglectus , Phlebotomus longiductus , Phlebotomus balcanicus and Sergentomyia minuta. Three of them ( P. perfiliewi , P. neglectus and P. balcanicus) were incriminated as vectors of L. infantum. Recent reports of autochthonous CanL in Romania require updates on sand fly distribution and diversity in this country. Methods Between 2013–2014 and 2016–2018, CDC light traps and mouth aspirators were used to collect sand flies in 132 locations from Romania, indoors and around various animal species shelters. Species identification of collected specimens was done using morphological keys, genetic tools and MALDI-TOF protein profiling. Results Sand flies were present in seven localities (5.3%): Eibenthal, Baia Nouă, Gura Văii (south-western Romania, Mehedinţi County); Fundătura, Pâhneşti, Epureni (eastern Romania, Vaslui County); and Schitu (southern Romania, Giurgiu County). Of the total number of collected sand flies ( n  = 251), 209 (83.27%) were Phlebotomus neglectus , 39 (15.53%) P. perfiliewi , 1 (0.40%) P. papatasi , 1 (0.40%) P. balcanicus and 1 (0.40%) P. sergenti (sensu lato). Conclusions We confirmed the presence of five sand fly species previously recorded in Romania. However, their updated distribution differs from historical data. The diversity of sand fly species in Romania and their presence in areas with Mediterranean climatic influences constitutes a threat for the reemergence of vector-borne diseases. In the context of CanL and VL reemergence in Romania, but also due to imported cases of the diseases in both humans and dogs, updates on vector distribution are imperative.

Background Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. Methods The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO 2 -CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO 2 -CDC traps were identified by morphological and molecular analysis. Results From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus ( n  = 273), Sergentomyia minuta ( n  = 5), P. mascittii ( n  = 2) and P. perfiliewi ( n  = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from −2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. Conclusions Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta , the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic. Graphical Abstract

Background Sand flies are vectors of the protozoan Leishmania spp. and phleboviruses. In Europe, several species are widely distributed in the Mediterranean region and a northward spread can be observed. They can be found regularly also in some regions of Central Europe, with Phlebotomus mascittii being the most cold-tolerant and northerly distributed species, but the knowledge on their distribution in countries such as Germany, Austria, Czechia, Slovakia, and Hungary remains fragmentary because of a lack of comprehensive field studies and a poor understanding of the ecological requirements and phylogeographic history. Methods A comprehensive literature review of sand fly occurrence in five Central European countries was complemented by entomological surveys, including sand fly and rodent screening for sand fly-borne pathogens. Nucleic acid extraction, COI barcoding, blood meal analysis, and phylogenetic and environmental analyses incorporating unsupervised machine learning techniques were conducted. Results This study significantly advances the understanding of the current distribution of six sand fly species in Central Europe. Among them, only Ph. mascittii was present in all analyzed countries, except Czechia, with its seasonal activity peaking in July. Phlebotomus papatasi , Ph. perfiliewi and Ph. neglectus were recorded in Hungary, while Ph. perniciosus and Phlebotomus simici were found in Germany and Austria, respectively. Although Leishmania DNA was absent in sand flies and rodents, DNA from two distinct Trypanosoma lineages was detected in several specimens, suggesting Ph. mascittii feeds on both birds and ruminants. Trypanosomatid lineages identified in local rodents differed, indicating distinct lineages between sand flies and rodents. Environmental analysis identified 15 Corine land cover classes associated with sand fly presence, with urban locations being the most frequently occupied. Linear regression models comparing presence versus absence revealed significantly higher sand fly presence in forested and urban landscapes. Furthermore, Ph. mascittii populations formed four distinct ecological clusters, which broadly grouped into two main geographic groups: one in the Upper Rhine Valley of southwestern Germany and the other spanning the Carpathian Basin. Conclusions This study provides new insights into the current distribution, ecological preferences, seasonal activity, and potential vector capacity of sand fly species in Central Europe. Graphical Abstract

Background Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus , which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis. Methods In the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS. Results In total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia ( Sergentomyia ) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri , suggesting that these vector species are opportunistic feeders. Conclusions Integrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp. Graphical Abstract

Background Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis. Since 2008, the Ministry of Health and the Ministry of Environmental Protection of Israel have conducted nationwide, periodic sand fly surveys. Initially, these surveys focused on localities with known endemic transmission of cutaneous leishmaniasis, but in recent years, entomological trapping has expanded to areas where prior information on sand flies was unavailable. Here we report the first confirmed occurrence of Phlebotomus (Larroussius) orientalis (Parrot, 1936) in Israel and place the Israeli material in a comparative phylogeographic context. Methods Entomological surveys by CO 2 trapping were conducted in the Negev Desert, southern Israel, between 2020 and 2024. Morphological sand fly identification was confirmed by sequencing fragments of the mitochondrial COI and Cytb / NADH1 genes. For a newly reported species, we inferred intraspecific phylogenetic relationships and divergence times between major clades. A subset of females was additionally screened for Leishmania DNA and vertebrate blood-meal sources by real-time polymerase chain reaction (PCR) coupled with high-resolution melt analysis. Results Targeted surveys and routine surveillance in the Negev region between 2020 and 2024 yielded 269 Phlebotomus orientalis (96 males, 173 females) among other species of local sand fly fauna from multiple wadi systems in the central Negev. These detections constitute the first confirmed records of Ph. orientalis in Israel. Species identification was confirmed through both morphological examination and molecular analyses of partial COI and Cytb / NADH1 genes. Phylogenetic analysis indicated that the Israeli Ph. orientalis specimens constitute a distinct lineage that diverged from East African conspecifics during the Early to Middle Pleistocene. Blood-meal analysis of engorged Ph. orientalis females identified the European hare as a vertebrate host, and none of the tested Ph. orientalis specimens were positive for Leishmania DNA. Conclusions Phlebotomus orientalis is a confirmed vector of L. donovani , the main agent of visceral leishmaniasis in East Africa. Its detection as a distinct and apparently long-established lineage in the Negev, in a region where parasites of the L. donovani complex are already involved in cutaneous leishmaniasis transmission, highlights the need to clarify the distribution, ecology, and host preferences of Ph. orientalis in Israel. Further studies are required to characterize its spatial and seasonal occurrence, evaluate its vector competence for L. donovani and L. infantum , and assess its potential contribution to current and future leishmaniasis transmission risks.

Background Leishmaniasis is a group of neglected vector-borne diseases transmitted by phlebotomine sand flies. Leishmania parasites must overcome various defenses in the sand fly midgut, including the insects’s immune response. Insect immunity is regulated by the ecdysone hormone, which binds to its nuclear receptor (EcR) and activates the transcription of genes involved in insect immunity. However, the role of ecdysone in sand fly immunity has never been studied. Phlebotomus perniciosus is a natural vector of Leishmania infantum ; here, we manipulated its neuroendocrine system using azadirachtin (Aza), a natural compound known to affect ecdysone synthesis. Methods Phlebotomus perniciosus larvae and adult females were fed on food containing either Aza alone or Aza plus ecdysone, and the effects on mortality and ecdysis were evaluated. Genes related to ecdysone signaling and immunity were identified in P. perniciosus , and the expression of antimicrobial peptides (AMPs), EcR , the ecdysone-induced genes Eip74EF and Eip75B , and the transcription factor serpent were analyzed using quantitative polymerase chain reaction (PCR). Results Aza treatment inhibited molting of first-instar (L1) larvae to L2, with only 10% of larvae molting compared to 95% in the control group. Serpent and Eip74EF , attacin, defensin 1, and defensin 2 genes were downregulated by Aza treatment in larvae. Similarly, Aza-treated adult females also presented suppression of ecdysone signaling-related genes and the AMPs attacin and defensin 2. Notably, all gene repression caused by Aza was reversed by adding ecdysone concomitantly with Aza to the larval or female food, indicating that these genes are effective markers for ecdysone repression. Conclusions These results highlight the critical role of ecdysone in regulating the development and immunity of P. perniciosus , which potentially could interfere with Leishmania infection. Graphical Abstract

Background. The wide distribution and high case-fatality ratio of severe fever with thrombocytopenia syndrome (SFTS) have made it a significant public health problem. This study was designed to identify the predictors of fatal outcomes and to evaluate the effectiveness of antiviral therapy in treating SFTS virus (SFTSV)–infected patients. Methods. A cross-sectional study was performed in a general hospital located in Xinyang city, whereas the largest number of patients with SFTS in China were treated during 2011–2012. The primary outcome for the treatment effect analysis was death. Other outcomes included sequential platelet levels and viral loads observed throughout the hospitalization and the interval between the initiation of ribavirin therapy and the return of the platelet count to a normal level. Results. A total of 311 SFTSV-infected patients were included in the study. The most frequent clinical presentations were fever, weakness, myalgia, and gastrointestinal symptoms. Each patient had thrombocytopenia, leukopenia, or both. The case-fatality ratio (CFR) was 17.4% (95% confidence interval [CI], 13.1%–21.6%). Older age (odds ratio [OR], 1.061; 95% CI, 1.023–1.099; P = .001), decreased level of consciousness (OR, 5.397; 95% CI, 2.660–10.948; P < .001), and elevated levels of lactate dehydrogenase (>1200 U/L; OR, 2.620; 95% CI, 1.073–6.399; P = .035) and creatine kinase (>800 U/L; OR, 2.328; 95% CI, 1.129–4.800; P = .022) were significantly associated with fatal outcome. The CFRs were similar between patients who received ribavirin and those who did not. Ribavirin treatment showed no significant effect on either platelet counts or viral loads during hospitalization of patients with fatal or nonfatal cases. Conclusions. These findings can improve knowledge about the characteristics of patients with fatal outcomes and the use of antiviral drug for SFTS.

Background An entomological study was conducted in the Canaraua Fetii Special Protection Area in the Dobrogea region, South-Eastern Romania. Four sand fly species were recorded at this location between 1968 and 1970: Phlebotomus neglectus , Ph . balcanicus , Ph . sergenti and Sergentomyia minuta . The most abundant sand fly species recorded at that time were Ph . balcanicus and Se . minuta . In the context of a countrywide study to update the sand fly species diversity, we surveyed the same area, recording also a previously unknown Ph . ( Transphlebotomus ) sp., for which we provide a formal description here. Methods Sand flies were collected between July and August in 2018 and 2019 in three sites from Canaraua Fetii, Dobrogea region, Romania. The general aspect of the landscape is of a canyon (vertical, narrow walls and deep valleys). Species identification was done using both morphological and molecular analyses. Results Out of 645 collected sand flies, 644 (99.8%) were morphologically identified as Ph . neglectus , while one female specimen (0.2%) was assigned to a previously unknown species, belonging to the subgenus Transphlebotomus . The morphological and molecular examination of this specimen showed that it is a previously unknown species which we formally describe here as Phlebotomus ( Transphlebotomus ) simonahalepae n. sp. Also, Ph . balcanicus , Ph . sergenti , and Se . minuta (previously recorded in this location) were not present. Conclusions The study revealed for the first time the presence of sand flies of the subgenus Transphlebotomus in Romania. Moreover, a new species, Ph . simonahalepae n. sp., was described based on a female specimen, raising the number of species in this subgenus to six. In the investigated natural habitat, the predominant species was Ph . neglectus instead of Ph . balcanicus and Se . minuta (recorded as the predominant species in 1968–1970). Graphical abstract

Background Leishmaniases are a group of medically and veterinary important diseases caused by protozoan parasites of the genus Leishmania (Kinetoplastida) and transmitted by blood-feeding female sand flies (Diptera: Phlebotominae). To assess the risk of Leishmania transmission, host exposure to sand fly bites can be measured through the detection of host antibodies to vector salivary proteins. Anti-sand fly saliva antibodies are elicited by repeated exposure of the mammalian host to sand fly salivary proteins deposited into the host skin during blood feeding. These antibodies are species-specific and correlate with the intensity of host exposure to sand fly bites. The aim of our study was to develop enzyme-linked immunosorbent assays (ELISAs) on the basis of recombinant sand fly salivary antigens as tools to measure exposure to sand fly bites, hence serving as risk markers for Leishmania transmission. We focused on two Old World vector sand fly species: Phlebotomus tobbi as a vector of Leishmania infantum , and Phlebotomus papatasi as a vector of Leishmania major . Methods Dog sera from endemic areas in Türkiye were used to characterise the main salivary antigens of P. papatasi and P. tobbi in immunoprecipitation and immunoblot assays, followed by proteomic analysis. Four candidate salivary proteins from each species were expressed in Escherichia coli and subsequently evaluated and validated in an ELISA as potential risk markers of dog exposure to sand flies. Results Among the eight tested recombinant candidates, P. tobbi rSP38 (a yellow-related protein), P. papatasi rSP36 (an apyrase) and P. papatasi rSP42 (a yellow-related protein) were identified as the most reliable antigens to replace salivary gland homogenate (SGH) in serological assays. They demonstrated high correlation with SGH and exhibited high sensitivity and specificity. Conclusions These recombinant antigens can be developed into a standardized assay to measure dog exposure to sand flies, which can serve as a complementing tool for leishmaniasis surveillance and control. Graphical Abstract