The Human Biting Culex pipiens Bioform molestus Detected in Several Areas in Southern Sweden

Introduction

Culex pipiens is a known vector for several pathogens, such as West Nile virus (WNV) and Usutu virus (USUV) (Brugman et al. 2018). The species is represented by two bioforms, Cx. pipiens f pipiens Linnaeus (L), 1758, and Cx. pipiens f molestus Forskal (F), 1775 (Diptera: Culicidae) (Harbach et al. 1985). In areas of sympatry, Cx. pipiens bioforms can interbreed to form hybrids. In northern Europe, they have separate but overlapping habitats and the hybridization rate between Cx. pipiens f pipiens and Cx. pipiens f molestus is lower than that in southern Europe where their habitats overlap more (Becker et al. 2012, Vogels et al. 2015, Brugman et al. 2018). It is of interest to distinguish Cx. pipiens f molestus from Cx. pipiens f pipiens that prefers avian hosts over mammals.

Culex pipiens is found in most parts of Sweden (Hesson et al. 2011). Culex pipiens f molestus has been found previously in Gothenburg and human biting Cx. pipiens has been reported in Stockholm in 1934, as reviewed in Lindström (2017). There are also findings of three Cx. pipiens f molestus from a farm and two wetlands outside Linköping in central south Sweden and hybrids in Linköping (Vogels et al. 2016). However, no other studies suggest that Cx. pipiens f molestus is present outside urban areas in Sweden.

To assess how widespread Cx. pipiens f molestus is in Sweden, we collected Culex mosquitoes through a citizen science outreach project from people in urban areas experiencing mosquito nuisance. Specimens were also sampled in three rural and periurban areas in the south of Sweden. We determined the specimens as Cx. pipiens using C oxidase subunit I (COI) and distinguished between the two bioforms, Cx. pipiens f pipiens and Cx. pipiens f molestus, by using the CQ11 microsatellite marker.

Materials and Methods

Mosquito samples were captured as part of a citizen science outreach project in Sweden, where people experiencing mosquito nuisance problems were encouraged to submit specimens. The information was disseminated through a press release from the National Veterinary Institute, SVA, and was picked up by several major news outlets (https://www.sva.se/aktuellt/pressmeddelanden/fanga-myggan-sva-s-insamling-pagar/). Citizens were instructed to collect blood-seeking mosquitoes when they landed on humans. This resulted in many mosquitoes sampled from indoor locations in both urban and rural areas, and sent between June and November during 2017 and 2018. The citizens collected one to seven mosquitoes in small containers, killed them by freezing, and sent them to SVA for identification.

In addition, mosquitoes were collected overnight in three areas in the south of Sweden using either Mosquito Magnet Patriot traps (Woodstream Corp.) that were run outdoors or using an exhaustor aspirator (Ento Sphinx s.r.o. CZ) that was run indoors. This was performed from June to September 2017, and in August 2018. From the collections, only mosquitoes morphologically identified as Cx. pipiens or Cx. torrentium, which are difficult to differentiate in adult females using keys (Becker et al. 2010), were included in the study. From the samples sent in by the public, some samples were in such a state that morphological identification was not possible. These specimens were nevertheless included.

DNA was extracted from two to three legs per mosquito and COI sequencing was performed as previously described (Lilja et al. 2017). The CQ11 assay was performed as described previously (Bahnck et al. 2006) to identify bioforms and hybrids but products were also purified, sent to Macrogen (Seoul, South Korea) and sequenced using the CQ11F2 primer. Sequences were compared to verify bioform classification.

Results

In total, there were 111 Culex mosquito specimens from southern Sweden, whereof 72 specimens were sent in by citizens and 39 were collected by research staff. Of these, 58 were defined as Cx. pipiens f molestus, 33 were Cx. pipiens f pipiens, 12 were Cx. torrentium, 1 was not a Culex mosquito (Aedes excrucians), 2 were hybrids of f pipens and f molestus, and 5 were unable to be classified due to a poor DNA quality. All Cx. pipiens f molestus mosquitoes were sent in by the public, and Cx. pipiens f pipiens and Cx. torrentium were uncommon in these samples (Supplementary Table S1). In Gothenburg (Fig. 1), Cx. pipiens f molestus was more spread throughout the city than previously reported and was also present in neighboring suburban locations. The molestus bioform was also present in the northern Malmö suburb Burlöv, and on two farms in rural locations dominated by farmland in Sollebrunn and Hörby. Culex pipiens hybrids were found in Simrishamn.

OPEN IN VIEWER

FIG. 1.

(A) Map showing the locations where Culex pipiens f molestus was found marked with red. Locations of former reports of Cx. pipiens f molestus are marked with black symbols. Historic presence is reviewed in Lindström (2017). Square: Stockholm (1941), pentagon: Lund (1933), triangle: Linköping (Vogels et al. 2016), and diamond: Gothenburg (Hesson et al. 2016). (B) The presence of Cx. pipiens f molestus in Gothenburg is shown in higher magnification. Color images are available online.

Discussion

Compared with the few previous reports of Cx. pipiens f molestus in Sweden, we have unexpectedly found specimens in several rural areas. Our study analyzed specimens sent in by people experiencing mosquito nuisance in the south of Sweden, allowing us to get samples that would be hard to obtain without citizen collaboration. Most of the Cx. pipiens f molestus specimens were collected in Gothenburg where the bioform has been recently described (Hesson et al. 2016). Although we cannot evaluate the true distribution of the molestus bioform, it is more common than previously thought. Our results indicated that the bioform may be identified as present in even more places, if investigated more closely.

In our study, we found Cx. pipiens f molestus only where people described nuisance problems indoors, and in these samples only three Cx. pipiens f pipiens were found. Although we did not have access to mosquito traps in these locations, it seems that passive surveillance using citizens who are urged to report indoor nuisance is a good method to find new populations of Cx. pipiens f molestus.

Our result contrasts with the results by Vogels et al. (2016), wherein they found Cx. pipiens f molestus in traps in rural sites and hybrids in an urban site where no nuisance problems have been reported. Further studies will be needed to more closely study the distribution of Cx. pipiens f molestus in Sweden and how this might affect future transmission of mosquito-borne viruses such as WNV and USUV that are spreading in Europe.

Conclusions

Culex pipiens f molestus was present in several urban and rural locations in southern Sweden and seemed to be associated with indoor nuisance problems.