Abstract Floodwater mosquito control in Sweden has been conducted since 2002, applying the larvicide VectoBac G® large granules by helicopter. The dosage has been gradually reduced from 15 kg/ha to 11 kg/ha, but a lower dosage than that was deemed too risky to achieve sufficient larval mortality in areas with patchy small water bodies. The small granulate variant of VectoBac G® consists of small even-sized granules with high bulk density. The number of granules per unit area is about 7.5 times higher than with the large granules which should allow for lower dosage with similar efficacy. We evaluated VectoBac G® small granules against floodwater mosquito larvae with 8 and 11 kg per hectare in a field test in four swamps and four wet meadows at Lake Färnebofjärden, Sweden, during a flood in May 2023. Dosage was randomly allocated to an equal number of each wetland type. In each study area, mosquito larvae were counted before and 24 hours after VectoBac G® small granules application. The mean number of mosquito larvae differed significantly before and after aerial application of small granules and total reduction of mosquito larval abundance was 92.6% with a dosage of 11 kg/ha and 98.5% with a dosage of 8 kg/ha. In the study areas with 8 kg/ha, larval abundance reduction was similar in wet meadows (98.8%) and swamps (98.1%). With 11 kg/ha, larval abundance reduction differed markedly between wet meadows (99.6%) and swamps (86.3%), due to one sample site in a swamp where a high number of alive larvae were found after application. This sample could be considered an outlier but it could also reflect natural conditions. We feel confident with the results and the usage of VectoBac G® small granules with a dosage of 8 kg/ha has been successfully implemented in regular treatments.

Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. In this work, we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground mosquito. Population genomic analysis of ~350 contemporary and historical samples counters the popular hypothesis that molestus originated belowground in London <200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Mediterranean or Middle East over the course of more than 1000 years, possibly in association with ancient agricultural civilizations of the Middle East. Our results highlight the role of early human society in priming taxa for contemporary urban evolution. They also provide insight into whether and how molestus contributes to West Nile virus transmission in modern cities.

This is the story of how an African virus unexpectedly turned up in a village in northern Europe and works of the people who have been involved in unraveling the mechanisms behind its movement and transmission. Dr. Jan Lundström started working with Sindbis virus as a student in the 1980s and is the main storyteller in this chapter. Dr. Jenny Hesson completed her PhD on the vectors of Sindbis virus in 2014, supervised by Dr. Lundström, and continues the investigation of this remarkable virus.

Floodwater mosquitoes are a main cause of mosquito nuisance in several European countries, including Sweden. Since 2002, a large-scale mosquito control programme around the river Dalälven in eastern Sweden targets floodwater mosquito species such as Aedes sticticus. The current study was initiated due to citizen complaints about nuisance mosquitoes around Deje village in Forshaga municipality, a region situated at the river Klarälven in western Sweden. Over three years, 2010-2012, mosquito collections with CDC-light traps and morphological species identification were performed to determine the nuisance causing mosquito species and to describe the seasonality, abundance and species composition of mosquitoes in the area. Over the three years of collections at eight trap sites, mosquito abundance increased with a mean of 282 mosquitoes collected per trap-night in 2010, 702 mosquitoes per trap-night in 2011, and 1,367 mosquitoes per trap-night in 2012. The increase in abundance was mainly due to an increase of floodwater mosquitoes, with Ae. sticticus as the main species. Single traps collected over 5,000 floodwater mosquitoes at several occasions in 2011 and 2012. A correlation between river discharge exceeding 300 m3/s and an increase in floodwater mosquito abundance three weeks later when average air temperatures were around 15 °C could be seen. Interestingly, river discharge was the highest in 2010, however mosquito abundance was higher in both 2011 and 2012. Potentially, the increasing number of diapausing eggs available after each season has contributed to the amplification of the Ae. sticticus population in the region.

The strategy of aerial control of the floodwater mosquito Aedes sticticus (Meigen) in the floodplains of River Dalalven, central Sweden, was developed to directly address specific larval breeding areas in temporary flooded wet meadows and swamps. Using the Bti-based larvicide VectoBac G (R), a very strong reduction of larval abundance is achieved, resulting in a massive decrease of blood-seeking females that could otherwise spread from the wetlands to feast on blood from humans and animals within 5 km or more from the larval biotopes. However, there is also a political demand to reduce the usage of the control agent through hypothetical alternatives, such as cattle grazing and mowing of the meadows, as well as hydrological changes of the River Dalalven. An evaluation of these measures showed that they are either insufficient or unrealistic in reducing floodwater mosquito abundance. Thus, we searched for other potential population suppression methods. Using the criteria of efficacy, environmental neutrality and compatibility within an integrated suppression approach, we conclude that Sterile Insect Technique (SIT) and the Incompatibility Insect Technique (IIT) would qualify for a pilot-scale test of their feasibility for the integrated control of the floodwater mosquito Ae. sticticus. The SIT and the IIT are similar strategies involving the release of sterile males which mate with local fertile females and result in infertile eggs. Prerequisites for a sterile male strategy to control Ae. sticticus include: a laboratory colony of the species, a facility for mass-rearing of mosquitoes, the sterilisation of males, a transport strategy, a dispersal system, assay systems for several life stages, and a method capable of reducing the population of this superabundant species before commencing the sterile male release. One factor in favour of implementing the SIT or IIT against Ae. sticticus is that mating occurs in or near well-defined larval breeding areas with specific relation to flood events. Another factor in favour of the SIT or the IIT is the availability of existing methods to measure gender, larvae and egg abundance. Also, existing Bti-treatments can substantially lower the population size before sterile male release. Other prerequisites, like the successful colonization of Ae. sticticus will require more tests and adaptations of existing mosquito rearing protocols. A pilot study is suggested for an isolated study area, protected from reinvasion by Ae. sticticus-females and included in routine Bti-treatments.

This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular.

Polyarthritis and rash caused by Sindbis virus (SINV), was first recognised in northern Europe about 50 years ago and is known as Ockelbo disease in Sweden and Pogosta disease in Finland. This mosquito-borne virus occurs mainly in tropical and sub-tropical countries, and in northern Europe it is suggested to cause regularly reoccurring outbreaks. Here a seven-year cycle of SINV outbreaks has been referred to in scientific papers, although the hypothesis is based solely on reported human cases. In the search for a more objective outbreak signal, we evaluated mosquito abundance and SINV prevalence in vector mosquitoes from an endemic area in central Sweden. Vector mosquitoes collected in the River Dalälven floodplains during the years before, during, and after the hypothesised 2002 outbreak year were assayed for virus on cell culture. Obtained isolates were partially sequenced, and the nucleotide sequences analysed using Bayesian maximum clade credibility and median joining network analysis. Only one SINV strain was recovered in 2001, and 4 strains in 2003, while 15 strains were recovered in 2002 with significantly increased infection rates in both the enzootic and the bridge-vectors. In 2002, the Maximum Likelihood Estimated infection rates were 10.0/1000 in the enzootic vectors Culex torrentium/pipiens, and 0.62/1000 in the bridge-vector Aedes cinereus, compared to 4.9/1000 and 0.0/1000 in 2001 and 0.0/1000 and 0.32/1000 in 2003 Sequence analysis showed that all isolates belonged to the SINV genotype I (SINV-I). The genetic analysis revealed local maintenance of four SINV-I clades in the River Dalälven floodplains over the years. Our findings suggest that increased SINV-I prevalence in vector mosquitoes constitutes the most valuable outbreak marker for further scrutinising the hypothesized seven-year cycle of SINV-I outbreaks and the mechanisms behind.

There are three human pathogenic bird-viruses transmitted by Culex mosquitoes in Europe: the alphavirus Sindbis and the flaviviruses West Nile virus and Usutu virus. Cases of Sindbis fever occur in the north while the flaviviruses are reported from southern Europe. In this study, 7933 Culex pipiens/torrentium mosquitoes from southern Sweden were screened by RTqPCR for these viruses. None of the mosquitoes were positive for viral RNA. The importance of mosquito species composition is discussed as a potential explanation to the lack of detection of mosquito-borne viruses in southern Sweden. However, continued surveillance of mosquitoes for Flaviviruses would be valuable as an early warning for public health awareness.

Major nuisance species are found among the floodwater mosquitoes and snow-pool mosquitoes, with the former being the main reason for mosquito control in most areas. Nuisance species vary with the area, and previous reports from northern areas conclude that the nuisance is most often caused by snow-pool mosquitoes. We investigated the mosquito fauna and abundances of host-seeking females using CDC traps baited with carbon dioxide, in Övertorneå city near the Arctic Circle in northern Sweden, after earlier complaints about massive mosquito nuisance. The abundance of host-seeking female mosquitoes was high in 2014, with a maximum of ∼15,400 individuals per CDC trap night, of which 89% was the floodwater mosquito Aedes rossicus. Surprisingly, the main nuisance species was a floodwater mosquito, occurring at the northernmost location it has ever been recorded in Sweden. Our report is probably the first documentation of such large numbers of Aedes rossicus in any locality and probably the first documentation of a severe floodwater mosquito nuisance near the Arctic Circle. Given the historical data on river discharge in the area, the nuisance is recurrent. We conclude that in northern localities, as well as in more southern localities, production of floodwater mosquitoes is a natural component of the floodplain fauna of rivers with a fluctuating water flow regime. Also, the floodwater mosquitoes Aedes sticticus and Aedes vexans were found north of their formerly known distribution in Sweden.

The floodplains of the River Dalälven, Central Sweden, have previously been known for enormous abundance of mosquitoes causing unbearable nuisance, but since mosquito control measures by Biologisk Myggkontroll (BMK) commenced in 2002, the area’s beautiful landscape and high biodiversity can be more appreciated. The floodplains are the central part of the UNESCO Nedre Dalälven River Landscape Biosphere Reserve as well as the location of various protected areas. The rich biodiversity also includes mosquitoes, although these insects are rarely considered in the study of biodiversity. We analysed mosquito data collected by the BMK surveillance program from 2001 until 2017 for species richness and assemblages of the mosquito fauna in the floodplains of the River Dalälven, and the nearby Creeks Vretaån and Kilsån. The mosquito species richness of the Nedre Dalälven River Landscape included 30 species in the present study, and three to eight species could potentially be added according to other studies, which then includes more than 70% of all known mosquito species in Sweden. The location of the floodplains at the Limes Norrlandicus, the border between southern and northern vegetation zones, is reflected in the mosquito fauna that includes species commonly found in Northern Sweden as well as more southern species. The mosquito species richness of the region is similar to the species richness observed in other floodplain areas in Central and Southern Europe. Hydrological conditions of the Lake basins of the River Dalälven floodplains varied from frequent flooding with high amplitude to almost no flooding. As expected, the hydrological variations were reflected in the mosquito assemblages, with high prevalence of floodwater mosquitoes in the flood-prone areas and more homogenous assemblages in the areas less subject to flooding. In conclusion, the River Dalälven floodplain area is considered a mosquito diversity hot-spot for Sweden and for Europe. Journal of the European Mosquito Control Association 36: 17-22, 2018

Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the Bacillus cereus group (Bcg) and has been used in biocontrol products against nematoceran larvae for several decades. However, knowledge is limited on whether long-term Bti application can affect the structure of indigenous communities of Bcg and the overall abundance of Bti. Using species- and group-specific quantitative PCR assays, we measured the Bcg- and Bti-abundances in riparian wetlands in the River Dalälven floodplains of central Sweden. On five occasions during one vegetative season, soil samples were collected in alder swamps and wet meadows which had been treated with Bti for mosquito larvae control during the preceding 11 years, as well as in untreated control sites and well-drained forests in the same area. The average abundance of Bcg in alder swamps was around three times higher than in wet meadows. Across all sites and habitats, the Bti treatments had no effect on the Bcg-abundance, whereas the Bti-abundance was significantly higher in the treated than in the control sites. However, for individual sampling sites, abundances of Bti and Bcg were not correlated with the number of Bti applications, indicating that added Bti possibly influenced the total population of Bti in the short term but had only a limited effect in the longer term. The findings of this study increase the understanding of the ecology of Bti applications for mosquito control, which can facilitate environmental risk assessment in connection with approval of microbiological control agents.

The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape genetics approach (FST , STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations, while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation-by-Distance patterns. Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low-to-moderate FST values on a broader geographical scale indicate a continuous between-population exchange of individuals, including considerable gene flow on the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed data considering population structure in the light of vector control strategies of the mosquito from public health importance.

Sindbis virus (SINV) is a mosquito-borne bird virus that occasionally causes human disease in Fennoscandia, suggested to have cyclic 7-year intervals between outbreaks. Reliable data on human infections in Sweden is however lacking. Here we investigated the SINV antibody prevalence among birds in a Swedish area endemic to SINV to scrutinize if a cyclic variation in antibody prevalence is present in the natural host of SINV. Serum from birds were sampled in the summers of 2002-2004 and 2009 in the floodplains of the River Dalälven in central Sweden, with 2002 and 2009 representing hypothesized years of SINV outbreaks. A total of 963 birds from 52 species (mainly passerines) were tested for the presence of SINV antibodies using a plaque reduction neutralization test. The highest SINV antibody prevalence was found in Turdidae species, specifically Fieldfare, Redwing and Song thrush in which more than 70% of sampled individuals had antibodies to SINV in 2009. The SINV antibody prevalence significantly varied between years with 2% in 2002, 8% in 2003, 14% in 2004 and 37% in 2009. Antibodies were found equally often in hatchlings and in adults and increased from early to late in the season. Clearly, the SINV antibody prevalence was not elevated in the bird hosts in the predicted outbreak year 2002, thus solid evidence of a cyclic occurrence of SINV in Sweden is still lacking.

Culex mosquitoes are vectors of several bird-hosted arboviruses that cause outbreaks in Europe, such as Sindbis virus and West Nile virus. Recently, the human-biting form of Culex pipiens, Cx. pipiens biotype molestus, was found causing big nuisance in a housing cooperative in Gothenburg in southern Sweden, confirmed by molecular identification. This is the first report of human-biting Culex in Scandinavia, signalling increased risk of arbovirus infection in northern Europe.

Mosquito species of the Culex genus are the enzootic vectors for several bird-associated viruses that cause disease in humans. In Europe, these viruses include Sindbis (SINV), West Nile and Usutu viruses. The morphologically similar females of Cx. torrentium and Cx. pipiens are potential vectors of these viruses, but difficulties in correctly identifying the mosquito species have caused confusion regarding their respective distribution, abundance, ecology, and consequently their importance as vectors. Species-specific knowledge from correctly identified field material is however of crucial importance since previous research shows that the relatively unknown Cx. torrentium is a far more efficient SINV vector than the widely recognized Cx. pipiens. The latter is involved in the transmission of several other viruses, but its potential importance for SINV transmission is debated.In this thesis I describe the development of a molecular method for species identification, based on reliably identified males of Cx. torrentium and Cx. pipiens. This identification method was then used in consecutive studies on the distribution and relative abundance of the two species in Sweden and 12 other European countries, SINV field infection rates in mosquitoes identified to species level, and evaluation of potential trap bias associated with common sampling techniques.The results showed that Cx. torrentium is a far more common species in Europe than previously assumed. In Sweden and Finland, it is the dominant species, accounting for 89% of the sampled Culex population. In central Europe, it is equally common to Cx. pipiens, while Cx. pipiens dominates south of the Alps Mountain range. Larvae of both species are often found together in both artificial containers (e.g. car tires) and natural sites. Also, a trapping bias against Cx. torrentium was revealed for CDC-traps. For the first time, SINV was isolated from species-identified Cx. torrentium and Cx. pipiens mosquitoes caught in the field, with Cx. torrentium being superior in infection rates (36/1,000 vs. 8.2/1,000). Future studies on SINV, as well as other mosquito-borne bird viruses in Europe, can hopefully gain from the baseline information provided here, and from principles of vector discrimination discussed in the thesis.

To investigate the effect of vegetation management on mosquito abundance on the floodplains of the River Dalälven, central Sweden, we studied abundance of floodwater mosquito larvae (mainly Aedes sticticus) using a paired design, comparing flooded wet meadows that were mowed or grazed by cattle to those that were unmanaged. Two floods occurred during the study year (2012), the first in conjunction with the spring flood in May and the second after heavy rainfall in July. We used the standard mosquito dipper to estimate mosquito larval abundance along transects from the shore towards permanent water on each meadow. The number of mosquito larvae was on average lower in each managed wet meadow compared to the corresponding unmanaged meadow, both in May (71 % reduction, SD = 28 %) and in July (35 % reduction, SD = 79 %). However, there was substantial variation in the difference between managed and unmanaged meadows among pairs. We measured water depth, temperature, estimated proportion of open water surface, soil nutrient levels and micro-topology. Some of the variation in larval abundance could be explained by differences in local conditions. Mosquito larval abundance increased with nutrient levels and occurrence of tussocks, and decreased with water depth and percentage open water surface, especially among unmanaged wetlands. This study suggests that mowing or grazing may reduce the abundance of floodwater mosquito larvae in flooded wet meadows. In addition, vegetation management seem to have the greatest effect on mosquito larval abundance in nutrient-rich wet meadows where tussocks are abundant.

We evaluate three trapping methods for their effectiveness at capturing Culex pipiens and Culex torrentium, both enzootic vectors of bird-associated viruses in Europe. The comparisons, performed in two regions in Sweden, were among CDC traps baited with carbon dioxide, gravid traps, and ovitraps baited with hay infusion. The proportions of the two Culex species in a catch differed between trap types, with CDC traps catching a lower proportion of Cx. torrentium than both gravid traps and ovitraps. Between gravid traps and ovitraps, there was no difference in the proportions of the two species. The results indicate that Cx. torrentium may go undetected or underestimated compared to Cx. pipiens when using carbon dioxide baited CDC traps. The new insight of trap bias presented here adds an important dimension to consider when investigating these vectors of bird-associated viruses in the field.

Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.

We isolated Sindbis virus (SINV) from the enzootic mosquito vectors Culex torrentium, Cx. pipiens, and Culiseta morsitans collected in an area of Sweden where SINV disease is endemic. The infection rate in Cx. torrentium mosquitoes was exceptionally high (36 infections/1,000 mosquitoes), defining Cx. torrentium as the main enzootic vector of SINV in Scandinavia.