A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

Diversity of mosquito fauna (Diptera, Culicidae) in higher-altitude regions of Croatia.

Global climate change and the accompanying rise in temperature could affect the biology and ecology of a number of vectors, including mosquitoes. High altitude areas that were previously unsuitable for the spread of mosquito vector populations could become suitable. The aim of this research was to study the distribution of mosquito species in higher altitude regions of Croatia. Samples were collected in three areas: Slavonian Mountains, Gorski Kotar, and Middle Velebit. Specimens were morphologically determined and confirmed by DNA barcoding and other genetic markers and showed the presence of 16 species belonging to six genera. The most abundant species were the Culex pipiens complex with 50% of the collected specimens. Both pipiens (Linnaeus, 1758) and molestus (Forskal, 1775) biotypes and their hybrids were identified within the complex, followed by Culex torrentium (Martini, 1925) (20.2%), Culiseta longiareolata (Macquart, 1838) (8.5%), and the invasive species Aedes japonicus (Theobald, 1901) (7.8% of the total number of collected specimens). The remaining 12 species made up 14.7% of the collected specimens. Intraspecific COI p-distances were within the standard barcoding threshold for OTUs, while interspecific genetic distances were much higher, confirming the existence of barcoding gaps. Mosquito fauna of Croatian mountains showed a moderate variety and made 30.8% of the total number of recorded mosquito species in Croatia thus far.