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The incidence of human and canine leishmaniasis in Europe is increasing and is a key indicator in the Lancet Countdown report on health and climate change 2024. While the potential distribution of the disease on a continental scale has been assessed under climate change scenarios, local analyses of vector species distribution are crucial for effective prevention strategies. Italy is endemic for Leishmania infantum , and expansions in the latitudinal and altitudinal distribution of canine cases and vector species have been recorded. This study evaluated the potential distribution of six phlebotomine sand fly species, known or suspected vectors of L. infantum , under climate change scenarios using ecological niche modeling and the maximum entropy (MaxEnt v. 3.4.1) modeling algorithm. We analyzed 410 records from 1979 to 2013 and projected future distributions for 2041–2060 and 2061–2080 using five CMIP5 models and two representative concentration pathways. Historical data from 1968 to 1972 were also examined to confirm suitable areas. The predictions indicate suitable habitats for sand flies throughout Italy, and variations among the different scenarios evaluated. All species show potential for expansion, such as the main vector, P. perniciosus , which shows an increase in the percentage of suitable habitat over time. On the other hand, predictions for P. perfiliewi don’t show a clear pattern, with an initial decrease and subsequent increase in suitable areas. Shifts in the distribution of insect vectors may affect the dynamics of Leishmania transmission , highlighting the need for improved surveillance strategies.

Background Sand flies (Diptera: Phlebotominae) are vectors of Leishmania spp., protozoan parasites that cause leishmaniasis, a zoonosis endemic in the Mediterranean region. Although Slovenia is not considered endemic, its proximity to affected areas and the presence of competent vectors underscore the importance of entomological surveillance. As part of the CLIMOS project, we investigated sand fly species composition, seasonal abundance, and the presence of Leishmania parasites at two sites in southwestern Slovenia: Cetore and Velike Žablje. Methods From May to October 2023, adult sand flies were collected using Centers for Disease Control and Prevention (CDC) miniature light traps. Specimens were morphologically identified and screened for Leishmania spp. DNA using quantitative polymerase chain reaction (PCR) targeted kinetoplast DNA, followed by species confirmation with ITS-1 nested PCR and restriction fragment length polymorphism (RFLP) analysis. Results A total of 274 sand flies were collected, predominantly Phlebotomus neglectus , P. perniciosus , and P. mascittii . Abundance peaked in July in Cetore and August in Velike Žablje, likely reflecting local climatic differences. Among 25 pools of female sand flies analyzed, Leishmania DNA was detected in three pools (12.0%), specifically in P. neglectus and P. mascittii . The cycle threshold values (Ct 35–37) indicated low parasite DNA loads. ITS-1 amplification was unsuccessful, likely due to low DNA concentration. Conclusions This study provides the first molecular evidence of Leishmania DNA in sand flies from Slovenia. The detection of Leishmania in P.  mascittii , a species with uncertain vector competence, is particularly noteworthy and warrants further investigation. Although species identification of the parasite was not possible, the findings suggest potential for autochthonous transmission and highlight the need for continued surveillance and research in the region. Graphical Abstract

Background Anopheles superpictus (subgenus Cellia ) plays an efficient role in malaria transmission in countries of the Mediterranean basin, Eastern Europe, and the Caucasus region, where it has been involved in the transmission of both Plasmodium falciparum and P. vivax . In Italy, this species was historically considered a secondary malaria vector, primarily recorded in the South including Sicily, along small rivers. It was less frequently observed in central Italy, only rarely reported in the North. Between 2022 and 2024, as part of routine investigations on residual anophelism, An. superpictus specimens were collected for the first time in the southern Maremma Plain (Magliano in Toscana, Grosseto, Tuscany). Methods Adult mosquitoes were collected in animal shelters and tool premises of two farms using traps and manual aspiration methods. Larval sampling was performed by exploring potential breeding sites along the Albegna riverbed. Anophelinae adults and larvae were morphologically identified at the species level, and subsamples were molecularly confirmed by sequencing the cytochrome c oxidase subunit 1 gene and the ribosomal DNA internal transcribed spacer 2 region. Results A total of 1,106 adult mosquitoes were collected from July to October 2022. Anopheles superpictus was the most abundant species recorded ( n  = 512; 46%), peaking in August and found in sympatry with An. labranchiae , the main historical malaria vector in Maremma Plain. Anopheles superpictus larvae were found in seven of the twelve investigated sites, with 50 specimens collected (mean density: 0.1–0.2 larvae per dip). Snapshot surveys in September 2023 and 2024 confirmed the stable occurrence of the species in the area. Conclusions Anopheles superpictus was consistently recorded in an area where it had not been previously documented. These findings provide new insights into the current presence and distribution of the species in Italy and at the European level, notably including the first recent identification of natural breeding sites. Furthermore, the results highlight the importance of residual anophelism surveys in malaria surveillance, supporting the development of targeted control strategies and preparedness plans for potential malaria reintroduction in at-risk regions. Graphical Abstract

Mosquitoes in the Culex pipiens complex are considered to be involved in the transmission of a range of pathogens, including West Nile virus (WNV). Although its taxonomic status is still debated, the complex includes species, both globally distributed or with a more limited distribution, morphologically similar and characterised by different physiological and behavioural traits, which affect their ability as vectors. In many European countries, Cx. pipiens and its sibling species Culex torrentium occur in sympatry, exhibiting similar bionomic and morphological characters, but only Cx. pipiens appears to play a vector role in WNV transmission. This species consists of two biotypes, pipiens and molestus, which can interbreed when in sympatry, and their hybrids can act as WNV-bridge vectors, due to intermediate ecological features. Considering the yearly WNV outbreaks since 2008 and given the morphological difficulties in recognising species and biotypes, our aim was to molecularly identify and characterised Cx. pipiens and Cx. torrentium in Italy, using recently developed molecular assays. Culex torrentium was not detected; as in other European countries, the pipiens and molestus biotypes were widely found in sympatry with hybrids in most environments. The UPGMA cluster analysis applied to CQ11 genotypic frequencies mainly revealed two groups of Cx. pipiens populations that differed in ecological features. The high propensity of the molestus biotype to exist in hypogean environments, where the habitat's physical characteristics hinder and preclude the gene flow, was shown. These results confirmed the CQ11 assay as a reliable diagnostic method, consistent with the ecological and physiological aspects of the populations analysed. Since the assessment of the actual role of three biotypes in the WNV circulation remains a crucial point to be elucidated, this extensive molecular screening of Cx. pipiens populations can provide new insights into the ecology of the species and may give useful indications to plan and implement WNV surveillance activities in Italy.

Background Among pathogenic Leishmania species, L. major and L. tropica , occur mainly in North African and Middle Eastern countries, primarily transmitted by Phlebotomus papatasi and P. sergenti respectively. In Italy, P. perfiliewi is the second most abundant sand fly species involved in L. infantum transmission exhibiting opportunistic behaviour in rural and semi-urban settings. Experimental infections were conducted to assess the susceptibility of a wild Italian P. perfiliewi population to the development of L. tropica and L. major . Methods Experiments were performed with a wild P. perfiliewi population, collected in Magliano in Toscana (Tuscany, Italy). In separate experiments, females were artificially infected with a suspension of L. tropica or L. major (10 6 promastigotes/ml). Infection progression was monitored through microscopic midgut dissection and supported by molecular analyses. Results Experimental infections with L. tropica yielded 5923 engorged females, with an average feeding rate of 25.7% and a survival rate of 70%. The infection positivity rate was 25.2%, significantly lower than that observed in the control species. Nonetheless, parasite migration to the stomodeal valve suggests that P. perfiliewi may support the full developmental cycle of L. tropica , although less efficiently than the control species. Infections with L. major showed a significantly lower mean feeding rate ( p  = 0.05) and a positivity rate of 46.6%, compared to 70.5% in P. perniciosus ( p  = 0.03). Still, P. perfiliewi appeared capable of supporting L. major metacyclogenesis, even under light to moderate parasite loads ( p  = 0.007). Conclusions These findings suggest that P. perfiliewi can support the development of both L. tropica and L. major under experimental conditions, with greater susceptibility observed for L. tropica . However, without in vivo confirmation or xenodiagnosis, its capacity for natural transmission remains unestablished. Nonetheless, implementing integrated One Health surveillance strategies would be advisable to monitor potential changes in the epidemiology of both exotic and endemic Leishmania species. Graphical Abstract

Background Phlebotomine sand flies (Diptera, Nematocera) are important vectors of several pathogens, including Leishmania parasites, causing serious diseases of humans and dogs. Despite their importance as disease vectors, most aspects of sand fly biology remain unknown including the molecular basis of their reproduction and sex determination, aspects also relevant for the development of novel vector control strategies. Results Using comparative genomics/transcriptomics data mining and transcriptional profiling, we identified the sex determining genes in phlebotomine sand flies and proposed the first model for the sex determination cascade of these insects. For all the genes identified, we produced manually curated gene models, developmental gene expression profile and performed evolutionary molecular analysis. We identified and characterized, for the first time in a Nematocera species, the transformer ( tra ) homolog which exhibits both conserved and novel features. The analysis of the tra locus in sand flies and its expression pattern suggest that this gene is able to autoregulate its own splicing, as observed in the fruit fly Ceratitis capitata and several other insect species. Conclusions Our results permit to fill the gap about sex determination in sand flies, contribute to a better understanding of this developmental pathway in Nematocera and open the way for the identification of sex determining orthologs in other species of this important Diptera sub-order. Furthermore, the sex determination genes identified in our work also provide the opportunity of future biotechnological applications to control natural population of sand flies, reducing their impact on public health.

Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling has emerged over the last decade as a method of choice for species identification of many medically important arthropods. However, the influence of intraspecific variability on the performance of this popular technique has seldom been tested. This study provides the first standardized comparison of different geographical populations of Phlebotomus perniciosus , a vector of Leishmania infantum and Toscana virus in the western Mediterranean, by MALDI-TOF MS protein profiling. Methods Phlebotomus perniciosus males were collected in five countries (Portugal, Spain, France, Italy, Croatia) that represent most of its distribution in Europe. All samples were trapped, stored and processed according to a highly standardized protocol to avoid effects other than geographical origin on their protein spectra acquired by MALDI-TOF MS protein profiling. The obtained protein spectra were compared with laboratory-reared specimens of Ph. perniciosus . Results Twenty-two analysed specimens from five geographical populations provided protein spectra that were highly similar, species-specific and clustering according to their quality. No grouping according to geographical origin was observed, and the protein spectra of field-collected specimens showed similar composition and complexity to spectra from Ph. perniciosus laboratory colony-reared in captivity for several decades. Conclusions Our findings demonstrate that in samples of a same sex, with the same collection method and storage time, MALDI-TOF MS protein profiling does not reflect the geographical origin of analysed specimens, confirming the value of this technique for high-fidelity and reproducible species identification of sand flies regardless of their geographical origin. Graphical Abstract

Background Sand fly-borne phleboviruses (SbPV) are globally distributed and pose potential public health risks. Despite increased detection in recent decades, detailed knowledge of their ecology, characteristics and clinical relevance remains limited. Many cases of SbPV infection likely go unreported or misdiagnosed due to limited awareness and the lack of standardized screening. The External Quality Assessment (EQA) reported herein was organized within the framework of the European Union CLIMOS (EU Climate Monitoring and Decision Support Framework for Sand Fly-borne Diseases Detection and Mitigation) project. The aim of this EQA was to standardize the detection of phleboviruses in order to provide comparable data to feed mathematical models for the surveillance of the impact of climate changes and environmental parameters on the kinetics and diversity of sand fly species and on sand fly-borne microorganisms. Methods Nine laboratories from seven countries participated in the EQA. Each laboratory was provided with eight vials, each containing an anonymous sample; two vials of lyophilized primers and probes to be used for the detection of Toscana virus (TOSV) and several Sandfly fever Sicilian virus (SFSV) species with a reverse-transcriptase PCR (RT-PCR) assay; and one vial of lyophilized primers for the detection of generic phleboviruses with a RT-PCR assay along with the standard operating procedure. The laboratories were instructed to submit their results together with details on the techniques employed. Results All nine laboratories successfully detected the two TOSV- and the one SFSV-positive samples. Only one laboratory, using a generic phlebovirus assay, detected all of the targeted phleboviruses. Conclusions All participating laboratories successfully identified the two TOSV and one SFSV using the proposed RT-qPCR assays, albeit with some variations in cycle threshold values across laboratories. The detection rate of SbPV was lower with the generic Phlebovirus assay than with the specific real-time RT-qPCR assays. This EQA aimed to assess the SbPV detection capabilities of molecular tools and strengthen their use, thereby supporting the involvement of laboratories in virus discovery and surveillance beyond their core expertise.

The incidence of visceral leishmaniasis (VL) in Albania is higher than in other countries of southern Europe, however the role of local sand fly species in the transmission of Leishmania infantum was not addressed conclusively. In 2006, a country-wide collection of sand flies performed in 14 sites selected based on recent occurrence of VL cases showed that Phlebotomus neglectus was by far the most prevalent species (95.6%). Furthermore, 15% of pools made from 422 P. neglectus females tested positive for Leishmania sp. genomic DNA. In the same year, Culicoides trapping was performed for bluetongue disease surveillance in 91 sites of southern Albania, targeting livestock farms regardless recent occurrence of VL in the surveyed areas. In 35 sites where sand flies were collected along with midges, Phlebotomus perfiliewi was the most prevalent among the Phlebotomus species identified, however search for leishmanial DNA in females of this species was unsuccessful. In 2011, sand flies were trapped in 4 sites of north Albania characterized by high VL incidence, and females were dissected to search for Leishmania infections. Both P. neglectus and P. tobbi were collected at high densities. Two positive specimens were detected from a sample of 64 P. neglectus trapped in one site (3.1%). Parasites were successfully cultured from one specimen and characterized as belonging to Leishmania infantum zymodeme MON-1, the only zymodeme so far identified as the agent of human and canine leishmaniasis in the country. Altogether our studies indicate that P. neglectus is the main leishmaniasis vector in Albania.

Background Dogs are the reservoir host of Leishmania infantum , the agent of zoonotic visceral leishmaniasis (VL), which is transmitted by the bite of phlebotomine sand flies. The sand fly Phlebotomus perniciosus is the main vector of zoonotic VL in the western Mediterranean region. Fluralaner has been shown to effectively kill this vector. The aim of this study was to evaluate the insecticidal efficacy of oral fluralaner in dogs bitten by P . perniciosus . Methods Two parallel-group, negative-controlled, randomized, masked laboratory trials with equivalent designs were performed in two different locations using two different pathogen-free laboratory-bred P. perniciosus strains for the challenge. In each trial, 12 purpose-bred beagles, initially ranked on natural attractiveness to sand flies, were randomly allocated to two groups (6 animals/group). Dogs in one group received fluralaner orally at the approved dose on day 0, and dogs in the control group were not treated. Each dog was subsequently exposed to an average of 70 unfed live sand fly females on days 1, 28, 56 and 84. Viability of blood-fed females was then evaluated for up to 96 h after exposure, and insecticidal efficacy was measured as the survival rate of flies fed on the fluralaner-treated dogs versus that of dogs in the control group. Significance was calculated for the proportion of live fed sand fly counts from treated versus control group dogs. Results Comparison of the survival proportions between treated and control groups showed that fluralaner insecticidal efficacy was highly significant in both trials ( P  < 0.001 or P  < 0.01 in different assessments) through to day 56. In the first trial, efficacy reached 100% on days 1 and 28, and 99.1% on day 56; in the second trial, the insecticidal efficacy was 98.5, 100 and 85.9%, respectively on the same days. On day 84, efficacy was in the range of 53–57% ( P  < 0.05) in the first trial and 0% in the second trial. Conclusion A single oral fluralaner administration to dogs under laboratory conditions results in strong and reproducible insecticidal efficacy against P. perniciosus for at least 8 weeks. Graphical Abstract