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Background Different trapping devices and attractants are used in the mosquito surveillance programs currently running in Europe. Most of these devices target vector species belonging to the genera Culex or Aedes, and no studies have yet evaluated the effectiveness of different trapping devices for the specific targeting of Anopheles mosquito species, which are potential vectors of malaria in Europe. This study aims to fill this gap in knowledge by comparing the performance of trapping methods that are commonly used in European mosquito surveillance programs for Culex and Aedes for the specific collection of adults of species of the Anopheles maculipennis complex. Methods The following combinations of traps and attractants were used: (i) BG-Sentinel 2 (BG trap) baited with a BG-Lure cartridge (BG + lure), (ii) BG trap baited with a BG-Lure cartridge and CO2 (BG + lure + CO2), (iii) Centers for Disease Control and Prevention-like trap (CDC trap) baited with CO2 (CDC + CO2), (iv) CDC trap used with light and baited with BG-Lure and CO2 (CDC light + lure + CO2). These combinations were compared in the field using a 4 × 4 Latin square study design. The trial was conducted in two sites in northeastern Italy in 2019. Anopheles species were identified morphologically and a sub-sample of An. maculipennis complex specimens were identified to species level by molecular analysis. Results Forty-eight collections were performed on 12 different trapping days at each site, and a total of 1721 An. maculipennis complex specimens were captured. The molecular analysis of a sub-sample comprising 254 specimens identified both Anopheles messeae/Anopheles daciae (n = 103) and Anopheles maculipennis sensu stricto (n = 8) at site 1, while at site 2 only An. messeae/An. daciae (n = 143) was found. The four trapping devices differed with respect to the number of An. messeae/An. daciae captured. More mosquitoes were caught by the BG trap when it was used with additional lures (i.e. BG + lure + CO2) than without the attractant, CO2 [ratioBG+lure vs BG+lure+CO2 = 0.206, 95% confidence interval (CI) 0.101–0.420, P 2 and CDC light + lure + CO2 (P = 0.321). The addition of CO2 to BG + lure increased the ability of this combination to capture An. messeae/An. daciae by a factor of 4.85, and it also trapped more mosquitoes of other, non-target species (Culex pipiens, ratioBG+lure vs BG+lure+CO2 = 0.119, 95% CI 0.056–0.250, P Ochlerotatus caspius, ratioBG+lure vs BG+lure+CO2 = 0.035, 95% CI 0.015–0.080, P Conclusions Our results show that both the BG-Sentinel and CDC trap can be used to effectively sample An. messeae/An. daciae, but that the combination of the BG-Sentinel trap with the BG-Lure and CO2 was the most effective means of achieving this. BG + lure + CO2 is considered the best combination for the routine monitoring of host-seeking An. maculipennis complex species such as An. messeae/An. daciae. The BG-Sentinel and CDC traps have value as alternative methods to human landing catches and manual aspiration for the standardized monitoring of Anopheles species in Europe. Graphical abstract

Mosquitoes (Diptera: Culicidae) were collected throughout Finland between 2013 and 2018 to determine species distributions. During the course of molecular identifications of specimens belonging to the Anopheles maculipennis complex, ribosomal internal transcribed spacer 2 sequences and link-reared specimens revealed the presence of Anopheles daciae Linton, Nicolescu & Harbach (n = 37), a new country record, as well as Anopheles messeae Falleroni (n = 19) in the collections. Although the sample size is low, distinctions are apparent in the distributions of these two species, with An. daciae present in south-eastern and central Finland, including the regions of Kanta-Hame, Pirkanmaa, Pohjois-Pohjanmaa, Paijat-Hame and Satakunta, and An. messeae present in the southern and south-eastern regions of Aland (Ahvenanmaa), Etela-Savo, Kanta-Hame, Kymenlaakso, Paijat-Hame, Satakunta, Uusimaa and Varsinais-Suomi. All reports of An. messeae in Finland prior to 2018 should therefore be recognized as potentially being either An. messeae or An. daciae. Because these species are potential vectors of malarial protozoa, it is important to have full knowledge of their distributions across Europe, particularly in the face of climate warming.

Four species of the Anopheles maculipennis complex have previously been recorded in Sweden. A recent addition to the complex is Anopheles daciae, which is considered to be closely related to, but distinct from Anopheles messeae. The original designation of An. daciae was based on five genetic differences (161, 165, 167, 362 and 382) in the second internal transcribed spacer (ITS) 2 of the ribosomal RNA. Further studies have shown that only two nucleotide differences (362 and 382) robustly separate the species. Thirty-three An. maculipennis complex mosquitoes were collected in the province of Uppland, Sweden. All were An. daciae but showed double peaks for three variable positions (161, 165 and 167). When cloned, the intra-individual nucleotide variation was almost exclusively fixed with either TTC or AAT, originally diagnostic for An. messae and An. daciae, respectively. To further investigate the intra-individual variation, nine An. daciae and 11 An. messeae were collected in southern Sweden and their ITS2 fragments were amplified and sequenced using Illumina MiSeq sequencing (Illumina, Inc., San Diego, CA, USA). For the diagnostic nucleotide 382 no intra-individual variation could be detected. However, although each An. daciae specimen carried several ITS2 sequence variants for the four other nucleotides, there was no intra-individual variation in the An. messeae specimens.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.