A snapshot mosquito field survey reveals the occurrence of Aedes geminus , Anopheles daciae , and Culex torrentium in the Republic of Ireland

A snapshot mosquito field survey was conducted in the Republic of Ireland in April–May 2017 and 2018 (f or the VectorNet project) to provide mosquito distribution data as there is a dearth of recent data available for that regio n. The occurrence data obtained serve as a baseline for a preliminary public and veterinary health risk assessment and to promote further targeted vector surveillanc e. Morphological identification was complemented by genetic analyses to determine the species identity of members of the Anopheles Maculipennis Complex and the Culex Pipiens Assemblage. A total of 52 samples, including 50 immature aquatic samples and 2 adult catches, were collected from 72 locations inspected and distributed over 30 of the 34 vice counties across the countr y. Seventeen mosquito taxa were collected, and here we provide the first records of Aedes geminus, Anopheles daciae and Culex torrentium for the Republic of Ireland. We also confirm the occurrence of both Anopheles algeriensis and Culiseta alaskaensis which have only been found once before in Ireland.


Introduction
Mosquito (Diptera: Culicidae) distribution data are crucial for informing and assessing public and veterinary risk related to pest or vector species, and they are relevant to understanding mosquito biology and diversity.
There is limited recent mosquito distribution data for the Republic of Ireland (hereafter Ireland).The National Biodiversity Data Centre, the Republic of Ireland, maintains a biodiversity data base with an online mapping tool.It has gathered 232 mosquito occurrence records collected between 1854 and 2008 for 18 species recorded from 122 locations in Ireland (NBDC, 2023).This data set consists of 97.8% of historical records from the period 1854-1991 (Ashe et al., 1991;IBS, 2023) and only 5 modern records (from 5 locations for the period 2001-2016) are listed.A rapid literature search on Web of Science™ on 26/III/2023 did not reveal additional records, and to our knowledge no recent mosquito survey of Ireland has been published.However, a recent distribution chart for Euro-mediterranean mosquito species lists 20 species for Ireland (Robert et al., 2019; Table 1).

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An. (Ano.)plumbeus Global change, in particular in land-use and climate, favours changes in distribution ranges for both native and invasive mosquito species (Elbers et al., 2015).This also applies to vector-borne diseases (VBDs) (Franklinos et al., 2019;Rocklöv and Dubrow, 2020) of which surveillance is better performed using an integrated approach (Braks et al., 2011).The One Health network 'VectorNet' was developed to apply this approach, commissioned by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC).VectorNet aims to facilitate risk assessments of VBD threats through the collection, mapping and sharing of distribution data for ticks, mosquitoes, sand flies, and Culicoides biting midges that are vectors of pathogens of importance to animal and/or human health in Europe (Braks et al., 2022).Capacity building and vector distribution data collections are among its foremost activities.Reporting presence or absence of any species is crucial to understand vector biology, biodiversity and distribution changes.Therefore, and because little modern mosquito distribution data exist for Ireland, we performed some capacity building on vector surveillance techniques together during the field surveys.Here we report the results of a snapshot mosquito field survey across the country performed in 2017 and 2018, as a baseline for a preliminary risk assessment and to enable further targeted vector surveillance.

Material and methods
A first survey was performed in 2017, from 15th to 18th of May (4 days), together with a tick survey and occasional trapping of Culicoides biting midges, along the western coast of Ireland.A second survey was performed in 2018, from 27th of April to the 10th of May (13 days), mainly over the central and eastern parts of the country.Habitats sampled were visually identified during road trips as suitable for mosquitoes (e.g.artificial containers, small ponds) or previously identified on satellite images (e.g.wetlands; Google Earth™ imagery).Mosquitoes were sampled according to standard procedures (Medlock et al., 2018).Mosquito immatures (eggs, larvae or pupae) were collected in aquatic habitats with an aquatic net and a white plastic tray, and specimens were removed from water with a pipette and placed in 70% ethanol in a plastic vial.Pupae and in some cases a few larvae (4th instar) were kept in water from breeding site until the adults emerged.Occasional adult collections were performed by human landing catches with a mouth aspirator and by resting catches with a sweep net (Bioform, Nürnberg, Germany) around human bait or vegetation.Larvae and adults were morphologically identified using available identification keys (Becker et al., 2010;Schaffner et al., 2001).Morphological identification was complemented by genetic assays to determine the species identity of members of the Anopheles Maculipennis Complex and the Culex Pipiens Assemblage.DNA isolation and amplification of the ribosomal internal transcribed spacer 2 (ITS2) was performed on a few Anopheles specimens, as described elsewhere (Collins and Paskewitz, 1996).The sequences obtained were compared with sequences in the GenBank database though the BLAST® online tool.To discriminate between Anopheles daciae and An.messeae, aligned sequences were visually checked for the presence of the five species-specific diagnostic polymorphic sites (Brusentsov et al., 2023;Nicolescu et al., 2004).The differentiation of the sibling species Culex pipiens and Cx.torrentium samples was performed by the multiplex PCR developed by Smith and Fonseca (2004).Briefly, the KAPA Express Extract Kit (KAPA-Biosystems, Wilmington, MA, USA) was used for DNA extraction from each specimen following the manufacturer's instructions.The multiplex PCR targeting the acetylcholinesterase-2 (ace-2) locus was carried out in 10 μl reactions, each one containing 5 μl Platinum Green PCR 2× mix (Invitrogen, Waltham, MA, USA), 0.3 pmol of each primer ACEquin, ACEtorr, ACEpip and B1246s (Smith and Fonseca, 2004), approximately 50 ng extracted DNA and ultra-pure water up to the volume of 10 μl.PCR conditions were as described in Smith and Fonseca (2004).

Results
Our snapshot mosquito field survey across Ireland resulted in 52 samples, including 50 immature samples and 2 adult catches (Table 2).A total of 72 locations distributed over 30 out of the 34 vice counties were investigated (Figure 1).Among them, 50 locations (69.5%) provided at least one mosquito specimen.
Overall, 1,959 mosquito specimens were collected, including 1,050 eggs, 630 larvae and 254 pupae in immature samples, and 23 females and 2 males in adult catches.An additional 151 females and 130 males were obtained from immature rearing to facilitate or confirm species identification.A total of 17 taxa were found.On five occasions the species could only be identified at the species group level (one larval sample of Ae. cinereus/geminus and four larval samples of Cx. pipiens/torrentium) (Supplementary Table S1).
Among the 18 species previously known to occur in Ireland (IBS, 2023), 14 species (78%) were observed.Two additional species, namely Ae. geminus and Cx.torrentium were also found.Our data are the first substantiated records for these species which were both included in Robert et al. (2019) based on our shared observations.Among the 24 specimens investigated by the multiplex PCR assay, only 17 resulted in a confident identification, explicitly 6 individuals of Cx. pipiens and 11 of Cx. torrentium.These specimens were obtained from two different locations for both species, without any sympatry observed.Larvae belonging to the Maculipennis Complex were found at one location, and ITS2 sequencing was conducted on 3 larvae and one adult male (obtained from a reared larva).Our sequences (GenBank  accession numbers PP263055-PP2630558) showed between 99.74 and 100% similarities with several sequences of both An.messeae and An.daciae.However, the visual check of the sequences aligned together with sequences from various countries allowed us to assign our four sequences to An. daciae, since three, four or all five species-specific diagnostic sites indicated that species (Figure 2).Among the 50 larval habitats which yielded mosquito specimens, a large majority were stagnant temporary water bodies (n=36; 72%) including ditches, ponds, dips in peat or forests, flooded forests and reed beds.The second most numerous were human-made (artificial) containers (n=8; 16%), such as diverse plastic containers, tyres, a discarded fridge and a wooden boat.The other sites were (semi-) permanent water bodies with vegetation (n=4; 8%), formed by borders of large ditches, ponds, and lakes, and slowly running water (n=2; 4%), in a ditch and a stream (Figure 3).

Discussion
A snapshot field study performed across the Republic of Ireland for 4 days in May 2017 and 13 days in April/May 2018 resulted in the detection of 17 mosquito taxa.Among these, 14 were already known to occur in Ireland, and three, namely Ae. geminus, An. daciae and Cx.torrentium are reported here for the first time.only be identified from each other morphologically by examining the male genitalia.Both species develop in natural temporary freshwater bodies between late spring and early autumn.We found specimens at two locations in North Kerry on 18 May 2017 (Figure 4A).In one case we morphologically identified Ae. geminus based on 2 male genitalia.Immatures of Ae. cinereus/geminus and Ae.geminus were collected from stagnant shaded pools, together with Ae. punctor in one case.There are four historical data under the name Ae. cinereus (1854-1985) (NBDC, 2023), with no indication of examination of male genitalia.Aedes (Ochlerotatus) cantans (Meigen, 1818) develops in natural temporary freshwater bodies in spring.We found it in Monaghan, Meath and Kilkenny, on 5 and 10 May 2018, in two forest ditches and a pond under willows.The identification of male genitalia (n=2, from one location) confirms the accuracy of the sorting from its sibling species Ae. (Och.)annulipes (Meigen, 1830).
Aedes (Och.)caspius (Pallas, 1771) and Aedes (Och.)dorsalis (Meigen, 1830) show morphological similarities and can be confused with each other.They develop in temporary brackish waters, thus mainly along the coasts, in spring and summer.We did not find them in our study.There is one historical record for Ae.caspius (1981), two for Ae.dorsalis (1927( and 1929( ) (NBDC, 2023)).
Aedes (Och.)punctor (Kirby, 1837) develops in temporary freshwater bodies, typically peat bogs, between spring and autumn.We collected specimens on one occasion in North Kerry on 18 May 2017, from a freshwater shaded pool (Figure 4A).There are only two historical records from 1970and 1985(NBDC, 2023)).
Aedes (Rusticoidus) rusticus (Rossi, 1790) develops in freshwater ponds in winter and spring.We collected it on five occasions in Monaghan ( 2 Anopheles (Anopheles) algeriensis Theobald, 1903 develops in temporary or semi-permanent freshwater bodies.We found specimens at two neighbouring locations in Offaly, on 6 and 7 May 2018 (Figure 4D).At the first site we were bitten by females at sunset, and at the second site, a few dozen metres away, we could collect larvae in a reed bed, together with An. claviger s.s. and Cs.morsitans.There is a single historical record (1989) from Clare (NBDC, 2023).
Anopheles  Anopheles (Ano.)maculipennis s.l.Meigen, 1818 and Anopheles (Ano.)daciae Linton, Nicolescu & Harbach, 2004: the first taxon includes undetermined species members of the Maculipennis Complex and the second is one of these members.Members of the complex can accurately be distinguished only by genetic analysis.They develop mainly in (semi-) permanent water bodies, from mid-summer to autumn.We collected specimens at one location in Galway, on 17 May 2017 from a pond (Figure 5D).There are nine historical records as An.maculipennis s.l.(NBDC, 2023), as no accurate molecular identification has been performed (Ashe et al., 1991).Some authors suggested An. (Ano.)messeae Falleroni, 1926 occurs in Ireland (Sinka et al., 2012;White, 1989) while others reported An. maculipennis s.s.Meigen, 1818 (Snow, 1998) or both of them together with An. (Ano.)atroparvus van Thiel, 1927(Wilkerson et al., 2021) to occur, but we did not find any substantiated record of these species in the literature.The four ITS2 sequences obtained from our specimens are all attributed to An. daciae despite some variability in the diagnostic nucleotide set (Figure 2).
Anopheles (Ano.)plumbeus Stephens, 1828 is a treehole breeding species, occasionally found in humanmade containers such as tyres, when the water is held for a long time.We caught one biting female in a forested area in Clare, 17 May 2017.There are also 10 historical records (1899-1990) (NBDC, 2023).
Coquillettidia (Coquillettidia) richiardii (Ficalbi, 1889) is a permanent waterbreeding species, of which the larvae and pupae breathe through the tissues of plant roots to which they are attached.Immatures develop in water bodies with erect vegetation, such as Phragmites, Typha, or Carex species.We did not detect this species in our field study, but there are two historical records (1926( and 1989( ) (NBDC, 2023)).
Culex (Culex) pipiens Linnaeus, 1758 and Culex (Cux.)torrentium Martini, 1925 are two sibling species that can be accurately distinguished only from examination of male genitalia.Culex pipiens itself is a species complex.In Europe, the complex comprises two forms (or biotypes) showing biological and behavioural differences and named pipiens and molestus.Among other traits, the first one is mainly ornithophilic, the second mainly mammalophilic.They develop in any artificial or natural water bodies, even with high organic matter (e.g.grey waters).In our field study we collected eight samples of specimens belonging to the species group (Table 3).While four samples could not be accurately identified to the species level, and thus are classified as Cx.pipiens/torrentium, three others were attributed to Cx. pipiens (Figures 5A,D) and three to Cx. torrentium (Figures 5A,B).Identification was performed by morphological examination of male genitalia for both Cx.pipiens (n=3) and Cx.torrentium (n=1) as well as on larvae submitted to the molecular assay (n=6 and 11, respectively, for two samples each).Both species were found sympatric in two samples.There are 78 historical records for Cx.pipiens (1832-1990), but there is no indication if male genitalia were examined, thus they should be referred to Cx. pipiens/torrentium (NBDC, 2023).
s.) Haliday, 1833 and Ae.(Och.)coluzzii Rioux, Guilvard & Pasteur, 1998 are two sibling species which can be distinguished only by molecular tools (protein electrophoresis or ITS2 sequencing).They develop in temporary brackish waters in spring and summer.We collected specimens at four locations in East Donegal (15 May 2017), West Cork (28 April 2018), West Galway (2 May 2018; Figure 4B) and Dublin (5 May 2018) in strandline pools, estuary puddles and a ditch, all flooded by brackish water.Since we could

Table 1
Summary of Culicidae species reported from Ireland by 2019.

Table 2
Sampling effort of our snapshot field study in the Republic ofIreland, 2017Ireland,  -2018.   .