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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

The Transphlebotomus subgenus has been drawing attention in last decade due to the uncertain vector capacity and cryptic life history of the group. During a 2015 entomological survey, Phlebotomine flies were collected in western locations of Slovenia, with a total of 22 specimens of the Transphlebotomus subgenus collected. In addition to morphological identifications, we confirmed the presence of Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) by nucleotide sequences of the mitochondrial cytochrome b gene. During the study, we found that P. mascittii is most abundant in the area of coastal–karst region of Slovenia, while in rare occasions can also be collected in Northeastern part of the country, near the Austrian border. Vector competence of P. mascitii, like all other member species of the Transphlebotomus subgenus, is unclear, but potential to transmit Leishmania (Kinetoplastida: Trypanosomatidae) pathogens or phleboviruses is strongly suspected.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arthropod-borne (arbo) viruses. While in Mediterranean parts of Europe the sand fly fauna is diverse, in Central European countries including Austria mainly Phlebotomus mascittii is found, an assumed but unproven vector of Leishmania infantum. To update the currently understudied sand fly distribution in Austria, a sand fly survey was performed and other entomological catches were screened for sand flies. Seven new trapping locations of Ph. mascittii are reported including the first record in Vienna, representing also one of the first findings of this species in a city. Morphological identification, supported by fluorescence microscopy, was confirmed by two molecular approaches, including sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) protein profiling. Sand fly occurrence and activity were evaluated based on surveyed locations, habitat requirements and climatic parameters. Moreover, a first comparison of European Ph. mascittii populations was made by two marker genes, cytochrome c oxidase subunit 1 (COI), and cytochrome b (cytb), as well as MALDI-TOF mass spectra. Our study provides new important records of Ph. mascittii in Austria and valuable data for prospective entomological surveys. MALDI-TOF MS protein profiling was shown to be a reliable tool for differentiation between sand fly species. Rising temperatures and globalization demand for regular entomological surveys to monitor changes in species distribution and composition. This is also important with respect to the possible vector competence of Ph. mascittii.

A large-scale entomological survey was carried out in summer 2016 in the Czech Republic and Slovakia. It revealed, for the first time, the presence of the phlebotomine sand fly Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Phlebotominae) in south-western Slovakia. Species identification of a captured female was confirmed by both morphological and sequencing (COI) analyses. Une enquête entomologique à grande échelle a été réalisée durant l'été 2016 en République Tchèque et en Slovaquie. Elle a révélé, pour la première fois, la présence de Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera : Phlebotominae) dans le sud-ouest de la Slovaquie. Une femelle a été capturée et son identification repose sur des caractères morphologiques et moléculaires (COI).