First mass development of Aedes albopictus (Diptera: Culicidae)-its surveillance and control in Germany.

The Asian tiger mosquito Aedes albopictus has undergone a dramatic expansion of its range in the last few decades. Since its first detection in 2007 in Germany at the motorway A5 coming from Italy via Switzerland to Germany, it has been continuously introduced by vehicles, most probably from Italy. After a hint from an alert gardener in an allotment garden area in Freiburg, Southwest Germany, in 2015, a surveillance programme was started focusing on the garden area and adjacent areas as well as most of the cemeteries as potential infestation areas. The surveillance programme confirmed a high infestation of the allotment garden. The container index (CI) exceeded almost 30% in August 2015. In lethal gravid Aedes traps (GATs) and BG-Sentinel traps, 4038 adults were caught. It could be proven that the Aedes population is more or less still spatially restricted to the allotment garden area which is adjacent to a train station where trucks from Novara, Italy, arrive loaded on trains. Outside the garden area, only a few breeding sites with developmental stages and adults were found within a radius of approximately 600 m from the highly infested garden area. It is most likely that Ae. albopictus females are constantly introduced as 'blind passengers' to Freiburg via trucks from Italy to Freiburg, Germany. After the first detection of the mass development of Ae. albopictus immediate and comprehensive control measures were initiated to reduce or even eliminate the Aedes population. Citizen awareness, especially of the gardeners, was increased by providing thorough information about the biology and control of Ae. albopictus. Beside environmental management, tablets based on Bacillus thuringiensis israelensis (Bti) were applied. The success of the control activities by the gardeners is reflected by the data gained during monthly inspection of the garden plots. The number of gardens without any container increased from 17% in July to 22% in August and 35% in September, 2015, resulting in a successful reduction of the Ae. albopictus population. The study underlines the importance of a comprehensive surveillance programme to assess the population density of Ae. albopictus as a basis for integrated control activities.

Successful overwintering of Aedes albopictus in Germany.

The Asian tiger mosquito (Aedes albopictus) is of great concern to public health authorities due to its vector competence and rapid spread across the globe. In 2015, two large local breeding populations of Ae. albopictus were discovered in southwest Germany. In spring 2016, we were able to demonstrate the first evidence of a successful overwintering in Germany of this originally tropical mosquito species in different research projects. Particularly noteworthy is the successful hatching of diapause eggs of an Italian strain (Calabria), which overwintered successfully in the field in St. Georgen im Schwarzwald (Baden-Wuerttemberg) at 820 m above sea level. Furthermore, within the scope of a larvae monitoring, the first larvae that hatched in the field were detected on the April 09, 2016 in a rain barrel within the Heidelberg population. Our first results show that self-extinction due to an unsuccessful overwintering cannot be assumed for populations of the Asian tiger mosquito which settled in Germany in previous years. The evidence of a successful overwintering of a large number of diapause eggs and the hatching of the first larvae in field conditions opens the control year against Ae. albopictus in southwest Germany.

The Culex pipiens complex in Europe.

The present study examines the mating and breeding behavior as well as the genetic differentiation of Culex pipiens biotype pipiens and Cx. pipiens biotype molestus. Firstly, the mating behavior of Cx. pipiens s.l. originating from larval populations of various epigeous and hypogeous breeding sites in Germany was examined. Autogeny was prevailing in underground populations, occasionally found in semi-open water reservoirs like drains, rarely in containers, but never in ponds and ditches. Secondly, in a multilocus enzyme electrophoretic study the gene flow among seven geographic populations of Cx. pipiens biotype pipiens and the biotype molestus from several European countries was quantified. For comparison, five populations of Cx. quinquefasciatus from Asia, Africa and North America, three populations of Cx. torrentium (Germany) and other outgroup species were also examined. Thirdly, the mitochondrial cytochrome c oxidase submit I gene of both biotypes from Germany was analysed by a polymerase chain reaction - restriction fragment length polymorphism assay and the ascertained DNA-sequences were aligned with genebank data of Russian populations. The population genetic analyses revealed much higher genetic distances between local populations of Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus compared to the low differentiation between geographically remote populations within each taxon. The UPGMA (unweighted pair group method with arithmetic mean) analysis and F-statistics positioned the biotypes pipiens on one side and molestus on the other side in discrete monophyletic clusters. Gene flow between local populations of the biotypes pipiens and molestus could be shown to be lower than gene flow between geographically distant populations within each of the two groups, leading to the conclusion that Cx. pipiens biotype molestus could be a distinct taxon. Culex quinquefasciatus was genetically well-separated, in particular by the diagnostic enzyme marker malate dehydrogenase (nicotinamide adenine dinucleotide phosphate). The genetic markers adenylatekinase and hydrobutyrate dehydrogenase allowed to screen thousands of morphologically similar samples of either Cx. pipiens s.l. and Cx torrentium and it could be shown that Cx. torrentium is a very frequent species in central Europe.

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing.

BACKGROUND: The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. METHODOLOGY/PRINCIPAL FINDINGS: To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

Microsatellite typing of Aedes albopictus (Diptera: Culicidae) populations from Germany suggests regular introductions.

The global spread of the Asian tiger mosquito Aedes albopictus is of concern, as this mosquito species constitutes an important vector of a number of emerging pathogens including dengue virus, chikungunya virus and Zika virus. Since its first appearance in Albania (1979) and Italy (1990), the species has been reported from more than twenty-five European countries. However, the dispersion process in Europe is largely unknown, as information on population genetic structure is lacking, which is relevant to understand the observed spread. In order to determine whether the ten Ae. albopictus populations detected in Germany until 2017 originate from a single introduction event or from independent importations, genetic analyses with a set of sixteen microsatellite markers were performed. The samples included specimens from three locations with potentially overwintering populations, collected in three consecutive years. The results indicate a heterogeneous population structure consisting of two clusters with significant substructuring, suggesting regular, independent introductions instead of a continuous spread across Germany originating from one or few sites. Moreover, the analyses provide further evidence for Ae. albopictus overwintering in Germany as samples from identical locations collected in three consecutive years had a relatively high genetic similarity. However, the population structure is probably influenced by local mosquito control activities. The results presented provide further evidence for regular introductions of Ae. albopictus specimens into Germany, probably leading to local establishment north of the Alps. This highlights the need for constant surveillance and control of Ae. albopictus not only in southern, but also in Central Europe.

Genetic differentiation of populations within the Culex pipiens complex and phylogeny of related species.

The Culex pipiens complex consists of several species, subspecies, forms, races, physiological variants, or biotypes according to different authors and includes the 2 holarctic variants Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. Differences in morphological characters are overlapping and thus are delimited in their taxonomic value, even when behavioral and reproductive specializations are apparent. Our enzyme electrophoretic study included 7 geographic populations of Cx. pipiens biotype pipiens and 7 of the biotype molestus from several European countries. For comparison, 5 populations of Culex quinquefasciatus from Asia, Africa, and North America were examined. The aim was an assessment of the extent of genetic differences between local populations of the biotypes pipiens and molestus versus the degree of differentiation between geographic populations of both groups. Culex torrentium, Cx. modestus, Culex stigmatosoma, and Culex territans were studied for comparison as taxonomical well-defined species. The population genetic analyses revealed much higher genetic distances between local populations of Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus compared to the low differentiation between geographic populations within each taxon. The UPGMA analysis and F-statistics position the geographic populations in discrete monophyletic clusters. Gene flow between local populations of the biotypes pipiens and molestus could be shown to be lower than gene flow between geographically distant populations within each of the 2 groups, leading to the conclusion that Cx. pipiens biotype molestus could be a distinct taxon. Culex quinquefasciatus could be diagnosed as genetically well separated, in particular by the diagnostic enzyme marker MDH (NADP). Two genetic enzyme markers were identified to differentiate Cx. torrentium from Cx. pipiens s.l. Culex modestus, Cx. stigmatosoma, and Cx. territans showed considerable genetic distances to the species of the Culex pipiens complex and between each other, and several genetic markers could be identified.

Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa.

UNLABELLED: Usutu virus (USUV), one of the most neglected Old World encephalitic flaviviruses, causes epizootics among wild and captive birds and sporadic infection in humans. The dynamics of USUV spread and evolution in its natural hosts are unknown. Here, we present the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution. The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow. Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks. Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles. IMPORTANCE: Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts.