DOI: 10.5061/dryad.zs7h44j5s
title: Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany

description: Accurate species identification is the prerequisite to assess the
 relevance of mosquito specimens, but is often hindered by missing or
 damaged morphological features. The present study analyses the
 applicability of wing geometric morphometrics as a low-cost and practical
 alternative to identify native mosquitoes in Germany. Wing pictures were
 collected for 502 female mosquitoes of five genera and 19 species from 80
 sampling sites. The reliable species identification based on interspecific
 wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross
 validation revealed an overall accuracy of 99% for the genus and 90% for
 the species identification. Misidentifications were mainly due to three
 pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes
 cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor.
 Cytochrome oxidase subunit I (COI) gene region was sequenced to validate
 the morphological and morphometric identification. Similar to the results
 of the morphometric analysis, the same problematic three Aedes-pairs
 clustered, but most other species could be well separated. Overall, our
 study underpins that morphometric wing analysis is a robust tool for
 reliable mosquito identification, which reach the accuracy of COI
 barcoding.
description: <b>Mosquito
 sampling</b> The right
 wings of 502 females of 19 mosquito species were analysed (Table 1).
 Mosquito specimens were collected at 80 study sites in Germany 2017 and
 2018 (Figure 1). Adults were sampled with two different methods:
 CO<sub>2</sub>-baited Biogents-traps (Biogents, Regensburg,
 Germany) or hand-made aspirators modified from Vazquez-Prokopec, et
 al.<sup>27</sup>. Immature stages were sampled with a standard
 dipper (Bioquip, CA, USA) in breeding sites and subsequently reared to
 adults in the laboratory. All specimens were identified by
 morphology<sup>17,28</sup> and stored at -18°C in a freezer
 until further analysis. Specimens of each species were selected from at
 least three different sampling sites (Table 1). The coordinates of the
 sampling location, sampling date and sampling method for each specimen are
 given in the supplementary material (Table S1). <b>Measuring wing shape</b> The right wing of each specimen was removed and mounted under a cover slip (15 <i>×</i> 15 mm) with Euparal (Carl Roth, Karlsruhe, Germany). Pictures of each wing were taken under 20<i>×</i> magnification with a stereomicroscope (Leica M205 C, Leica Microsystems, Wetzlar, Germany). Fiji<sup>29</sup> as bioscience package of ImageJ<sup>30</sup> was used to digitize 18 landmarks (Figure S1). The landmark selection was in accordance with other studies analysing mosquito wing morphometry<sup>21-23,31</sup>. The wing pictures were divided among two observers (authors LE and FGS) and digitalised in random order to minimize a memory biased landmark collection between the mosquito specimens of the same species. One month later, the measurement was repeated for three specimens per species by four observers (authors LE, LJ, RL and FGS) to assess the degree of observer error and repeatability in landmark collection<sup>32</sup>. <b>Genetic identification </b> DNA isolation was performed from the whole mosquito body, except of the right wing. Individual specimens were placed into 2 ml tubes and about 10 pieces of 2.0 mm zirconia beads (BioSpec Products, Bartlesville, USA) as well as 1 ml of cell culture medium (high-glucose Dulbecco’s modified Eagle’s medium; Sigma-Aldrich, St. Louis, MO, USA) were added. The homogenization was performed with a Tissuelyser II (Qiagen, Hilden, Germany) for 2 min at 30 oscillations/s and 200 μl of the homogenate were used for DNA extraction, which was performed with KingFisher™ Flex Magnetic Particle Processor using MagMAX™ CORE Nucleic Acid Purification Kit (both Thermo Fisher Scientific, Waltham, MA, USA). Polymerase chain reaction (PCR) amplification of cytochrome oxidase subunit I (COI) gene region was conducted with the protocol published by Fang, et al.<sup>33</sup> using the primers by Folmer, et al.<sup>34</sup>. Sanger sequencing was applied for all positive amplicons (LGC Genomics, Berlin, Germany). Furthermore, morphologically identified <i>Cx. pipiens</i> s.l. and <i>An. maculipennis</i> s.l. specimens were typed to species level (<i>Cx. pipiens pipiens </i>form <i>pipiens</i> resp. <i>Anopheles messeae</i>) using two molecular assays<sup>35,36</sup>.
description: Herein, we provide all mosquito wing pictures used in this study,
 including metadata on the sampling location, date of sampling and sampling
 method for each specimen.