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Additional file 5: Figure S1. Haplotype networks constructed for P. balcanicus from Romania (GenBank Accession Number: MK425636), Serbia (GenBank Accession Number: MN003380), Turkey (GenBank Accession Numbers: MN086653- MN086654) and P. creticus from Crete (GenBank Accession Numbers: MT501623-MT501638). The relative frequency of haplotypes was reflected by the size of the circle; missing haplotypes were illustrated by the small circles, and dashes represent the mutational steps.

Additional file 2: Figure S1. Haplotype networks obtained for the members of Transphlebotomus subgenus (a Cytb; b CO1; c ND4). The sequences of Ph. anatolicus, Ph. canaaniticus, Ph. economidesi, Ph. killicki, and Ph. mascittii were obtained from GenBank (AY780350, KF483664, KR336642-336659, KX869078, KX963380, KY848831, MN003381, MN812827-MN812830, MT332686–MT332688). Haplotypes are sized according to their relative frequencies. Different colors represent different species and black-filled circles represent missing haplotypes. The number of mutational steps are indicated by the dashes.

Additional file 1: Table S1. Trapping data included in the study with the recorded sand fly composition, Canaraua Fetii, Dobrogea Region.

Additional file 1: Table S1. Historical data review on the sand fly fauna of the study area. Table summarizing essential published literature between 1910 and 2019.

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe., Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction. Combining niche distribution modeling, genotype–environment associations and generalized dissimilarity modeling, we found evidence for environmental‐induced adaptive changes in the Asian tiger mosquito Aedes albopictus after its introduction in Europe. The ability to rapidly evolve observed in invasive populations together with a large proportion of unfilled potential suitable areas pave the way to further spread of Ae. albopictus in Europe.

VectorNet (the European network for sharing data on the geographical distribution of arthropod vectors, transmitting human and animal diseases) started mid-2014 as the successor of VBORNET and is funded by both ECDC (the European Centre for Disease Prevention and Control) and EFSA (European Food Safety Organisation). The network aims at bringing together both medical and veterinary entomologist as decision-makers in the public and animal health domain. It not only gathers published data on the distribution of vectors but actively collects data in the field (about 2/3th of the entire budget). By doing so the network focus on capacity building, strengthening of collaborations and the standardisation of data collection at the Pan-European level. After 3 years, a huge effort has been made and numerous field activities has taken place across Europe, setting the standards for future work. Besides all these activities, the network has assisted the funding agencies by providing scientific support for their opinion reports and requests from the European Commission. This presentation will deal with the past, ongoing and future activities, pitfalls and opportunities of such a network with focus on the major achievements. (Texte integral)

Bloodsucking females of phlebotomine sand-flies are the only proven vectors of Leishmania parasites, causative agents of leishmaniases. Their conclusive species identification in endemic areas, where morphologically similar species with different vectorial capacity and ecology occur, is profound. Traditional morphological approach relies on minute and sometime dubious characters ; therefore several molecular methods were applied recently. Protein profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a promising technique for identification of various organisms including insects. It is simple, accurate, and requires minimal sample preparation. The obtained mass spectrum represents a characteristic and unique protein fingerprint, which allows rapid and effective species identification. We optimized methods of specimen capture, storage, sample preparation, tested suitability of different body parts of sand fly for protein identification, and developed a multi-approach protocol that utilizes a single insect body for several identification methods (morphological, DNA and protein based). Further on, the influence of blood meal at several time intervals after feeding on the quality of protein profile of females was investigated. Also spectra of larvae (L4) and pupae were measured and compared with profiles of adult sand flies. A reference spectra database comprising 16 sand fly species was established using MALDI Biotyper software. It includes species maintained in laboratory colonies as well as field-collected specimens identified by morphological analysis and DNA sequencing. During two field trips to Balkan countries (Bosnia and Hercegovina, Bulgaria, Croatia, Hungary, Macedonia, Montenegro, Serbia and Slovenia) in summer 2015, 680 specimens (Larroussius, Adlerius, Paraphlebotomus, Phlebotomus, Sergentomyia) out of 7257 totally captured were identified by protein profiling which proved as reliable. It was useful for female specimens of the subgenus Larroussius (492 analyzed specimens) and differentiation between its closely related species (P. neglectus, P. perfiliewi, P. tobbi). Also two species of the subgenus Adlerius (P. balcanicus, P. simici), hardly distinguishable by morphology, were unambiguously differentiated by protein spectra. All dubious samples (difference between morphological and protein based ID) were clarified by a sequencing analysis, which confirmed the identification according to MALDI-TOF MS in all cases. In summary, MALDI-TOF protein profiling is a suitable, time- and cost- effective method for species identification of large sets of field-caught sand flies if these are collected, stored and analyzed using a standard, optimized protocol.

Conclusive species identification of sand flies by morphological analysis remains a challenging task due to limited availability of robust characters, their intraspecific variability among different populations and a need of expertise to assess them. According to proposed scenarios of future climatic trends, sand flies may emerge in regions with a lack of expertise for their species identification by classical morphological approach. Therefore, especially for high-throughput assessment of field surveys in endemic areas, there is a need for complementary methods of species identification relying on molecular techniques. Protein profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry emerged as a molecular method recently used for species identification of various medically important arthropod vectors including phlebotomine sand flies. With a standardized protocol of specimen capture, storage and sample preparation and a robust reference database, it provides characteristic and unique protein fingerprints that allow conclusive species identification. It requires minimal sample preparation that enables utilization of a single sand fly specimen for several purposes. Balkan countries are in a transition area in the European part of the Mediterranean basin where sand fly fauna of the western part meets with eastern elements and where several species have the limit of their geographical distribution. At the same time, data on sand fly presence and species composition are scarce due to recent upheavals. Due to two field surveys in the Balkan area and adjacent countries (Bosnia and Hercegovina, Bulgaria, Croatia, Hungary, Macedonia, Montenegro, Serbia and Slovenia) in summer of 2015, approx. 700 sand fly specimens of a total of 8000 captured were analyzed by protein profiling. The sand flies were trapped by CDC traps placed in domestic, peridomestic and sylvatic sites and processed according to standardized protocol. An in-house reference spectrum database was used as reference comprising 16 sand fly species based on specimens from both laboratory colonies as well as field-collected specimens whose species identity was conclusively confirmed by morphological analysis and DNA sequencing. Analyzed specimens belonged to species of four subgenera of the genus Phlebotomus : Adlerius ( Ph. balcanicus and Ph. simici ), Larroussius ( Ph. neglectus , Ph. perfiliewi , Ph. tobbi ), Paraphlebotomus ( Ph. sergenti )and Phlebotomus ( Ph. papatasi ). Moreover, two species of the genus Sergentomyia were also recorded ( Se. minuta and Se. dentata ). The method proved very suitable for identification of female specimens of the subgenus Larroussius (492 analyzed specimens) where species-specific diagnostic markers were characterized beside the overall protein spectra. All dubious specimens where morphological and protein-based identification did not agree were further characterized by a sequencing analysis (cytochrome oxidase 1 gene) which confirmed the identification by MALDI-TOF MS in all cases. MALDI-TOF protein profiling proved to be a time- and cost-effective method of choice for species identification of large sets of field-caught sand flies if these are collected, stored and analyzed using a standard, optimized protocol. Financial support: This project is supported by the Czech Science Foundation (GA15-04329S) and by VectorNet.

VectorNet ‘‘A European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents’’ project is supported by the EU-ECDC/EFSA consortium and coordinated by Avia-GIS, Belgium. This study shows some of the results and achievements of the sand fly-team efforts in eight Balkan countries in 2015 in the framework of the VectorNet Project. Eight countries (Bosnia & Herzegovina, Montenegro, Croatia, Bulgaria, Macedonia, Serbia, Kosovo, Slovenia), including 267 locations and 36 cities were studied by the sand fly team with the aim of determining the altitudinal and trans-sectional distribution of species, identifying species and detecting possible pathogens in the Balkans. Sand flies were collected with light traps for a total of 951 trap nights during the field missions. From this study, 12 species were identified and a total of 9, 096 specimens collected from fieldwork. The results show that Phlebotomus neglectus (74%) is the dominant species in Balkan countries and this species was collected from all eight countries together with Ph. tobbi (10%). These two species comprise 84% of total sand fly abundance. Other species include: Ph. perfiliewi s.l (6.13%), Sergentomyia minuta (3.56%), Ph. perniciosus (1.57%), Ph. papatasi (1.35%), Ph. simici (0.9%), Ph. mascitti (0.45%), Ph. sergenti (0.1%), Ph. alexandri (0.07%), and Ph. balcanicus (0.03%) (found only in Montenegro). We also calculated some of the important community parameters such as similarity, richness, species diversity, species evenness and dominance for each country. The most similar countries in terms of species composition are B&H-Kosovo (0.77), Bulgaria-Kosovo (0.85), Serbia-Kosovo (0.86), Croatia-Macedonia (0.80) and Bulgaria-Macedonia (0.80). From these results, it appears that Macedonia and Serbia are ecotones (transition areas) in the Balkans. The highest species diversities were observed in Macedonia (1.366), Bulgaria (1.247) and Serbia (1.169), respectively. The lowest was B&H with 0.386 values. In contrast, B&H, like other less diverse countries, has the highest dominance value (81.4%). In pathogen detection studies, two novel viruses in B&H and Macedonia and Leishmania infantum parasites in B&H, Macedonia and Kosovo were detected. To the best of our knowledge, most of the information derived from this study was new to the Balkan countries.