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

Abstract

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.

Keywords: Cryptic species.; Culicidae.; Cytochrome c oxidase I (COI).; Internal transcribed spacer 2 (ITS2).; Invasive species.; Species complex.; Species delimitation.; Species identification..

Fig. 1

Sampling localities in ​​three biogeographic regions of Croatia: Pannonian-Peripannonian region—green dots, Mediterranean region—blue dots, and mountainous region—orange dots; high-altitude localities in the Pannonian-Peripannonian region are also marked with orange dots. Some localities may represent multiple sampling sites (details for each sample are available in the public BOLD dataset DS-CROCU2)

Fig. 2

Frequency histogram of p-distances within and between investigated mosquito species (MOTUs) for the public BOLD dataset DS-CROCU2

Fig. 3

ML tree based on COI DNA sequences of the specimens sequenced in this study. Numbers on branches denote bootstrap / aLRT support values (values lower than 70% are not shown). The results of species delimitation methods (bPTP, ASAP and BIN-RESL) are shown as vertical bars on the right

Fig. 3

ML tree based on COI DNA sequences of the specimens sequenced in this study. Numbers on branches denote bootstrap / aLRT support values (values lower than 70% are not shown). The results of species delimitation methods (bPTP, ASAP and BIN-RESL) are shown as vertical bars on the right