Recently discovered Aedes japonicus japonicus (Diptera: Culicidae) populations in The Netherlands and northern Germany resulted from a new introduction event and from a split from an existing population

BACKGROUND: Originally native to East Asia, Aedes japonicus japonicus, a potential vector of several arboviruses, has become one of the most invasive mosquito species in the world. After having established in the USA, it is now spreading in Europe, with new populations emerging. In contrast to the USA, the introduction pathways and modes of dispersal in Europe are largely obscure. METHODS: To find out if two recently detected populations of Ae. j. japonicus in The Netherlands and northern Germany go back to new importations or to movements within Europe, the genetic makeup of mosquito specimens from all known European populations was compared. For this purpose, seven microsatellite loci from a representative number of mosquito specimens were genotyped and part of their mitochondrial nad4 gene sequenced. RESULTS: A novel nad4 haplotype found in the newly discovered Dutch population of Ae. j. japonicus suggests that this population is not closely related to the other European populations but has emanated from a further introduction event. With five nad4 haplotypes, the Dutch population also shows a very high genetic diversity indicating that either the founder population was very large or multiple introductions took place. By contrast, the recently detected North German population could be clearly assigned to one of the two previously determined European Ae. j. japonicus microsatellite genotypes and shows nad4 haplotypes that are known from West Germany. CONCLUSION: As the European populations of Ae. j. japonicus are geographically separated but genetically mixed, their establishment must be attributed to passive transportation. In addition to intercontinental shipment, it can be assumed that human activities are also responsible for medium- and short-distance overland spread. A better understanding of the processes underlying the introduction and spread of this invasive species will help to increase public awareness of the human-mediated displacement of mosquitoes and to find strategies to avoid it

Population genetics and distribution of the invasive mosquito Aedes japonicus japonicus (Diptera: Culicidae) in Germany and Europe

Die vorliegende Arbeit befasst sich mit der Verbreitung und Populationsgenetik der invasiven asiatischen Buschmücke Ae. j. japonicus, die sich seit 2008 durch Menschen-vermittelten Transport in Deutschland ausbreitet. Aedes j. japonicus ist unter Laborbedingungen Vektor für verschiedene Viren, unter anderem für das Dengue-Virus und das Chikungunya-Virus, und wurde im Feld mit dem Japanische Enzephalitis-Virus, dem West Nil-Virus und dem La Crosse-Virus infiziert gefunden. 2012 wurde aufgrund mehrerer unabhängiger Mücken-Einsendungen im Rahmen des Citizen-Science-Projekts “Mückenatlas” eine Population der Asiatischen Buschmücke in Westdeutschland entdeckt. Das Verbreitungsgebiet dieser Population befand sich weit nördlich der bisher angenommenen nördlichen Verbreitungsgrenze der Art in Süddeutschland. Das Ausmaß der Population wurde nach einem zeitlich begrenzten Monitoring auf eine Fläche von ca. 2000 km2 bestimmt. Aus dieser Population wurden Individuen von fünf Orten populationsgenetischen Analysen unterzogen, um verwandtschaftliche Beziehungen innerhalb der Population und im Vergleich zu anderen europäischen Populationen aufzudecken. Hierzu wurden sieben Mikrosatelliten-Loci untersucht. Zusätzlich wurde ein Teil der mitochondrialen nad4-Genregion der Individuen auf Nukleotid-Polymorphismen untersucht. Die Ergebnisse wurden mit bereits zuvor erhobenen Daten von Populationen aus der Schweiz, aus Österreich/Slowenien und Belgien verglichen. Die Mikrosatellitensignatur der westdeutschen Population unterschied sich deutlich von der der anderen europäischen Populationen. Weiterhin wurden verschiedene nad4-Haplotypen gefunden, die zuvor nirgendwo sonst in Europa aufgetreten waren. Demnach ist zu vermuten, dass diese Population auf eine unabhängige Einschleppung von Individuen aus Übersee zurückgeht. Der genaue Ursprung – USA oder Ostasien – konnte nicht bestimmt werden. 2013 wurden zwei weitere Ae. j. japonicus-Populationen in Europa entdeckt: eine in Norddeutschland und eine weitere in den Niederlanden. Die genetischen Signaturen von Individuen dieser Populationen wurden wie beschrieben analysiert. Zusätzlich wurde das genetische Material einer größeren Menge von Individuen aus Slowenien sowie von Individuen aus Kroatien und Süddeutschland untersucht. Die Ergebnisse wurden mit denen aus der vorigen Studie verglichen und zeigten aufgrund einer ähnlichen Mikrosatellitensignatur und gleicher nad4-Haplotypen klar, dass die norddeutsche Population eine Subpopulation der westdeutschen ist. Die geringe Populationsdichte und die vergleichsweise kleine Ausdehnung der norddeutschen Population deuten außerdem darauf hin, dass die Abspaltung nicht lange zurückliegt. Die niederländische Population scheint hingegen auf einer weiteren Einschleppung von Individuen aus Übersee zu basieren. Im Spätsommer 2015 wurde die bisher letzte deutsche Ae. j. japonicus-Population in Oberbayern und dem angrenzenden Österreich entdeckt. Populationsgenetischen Analysen zufolge ist diese Population eng mit der früher beschriebenen österreichisch-slowenischen Population verwandt und unterscheidet sich von allen anderen deutschen Populationen, was darauf schließen lässt, dass es sich bei ihr um eine Abspaltung von der österreichisch-slowenischen Population handelt. Die Ver- und Ausbreitung von Ae. j. japonicus in West- und Norddeutschland wurde vom Zeitpunkt der Entdeckung in 2012/2013 bis 2015 beobachtet. In dieser Periode erweiterte die westdeutsche Population ihr Verbreitungsgebiet beträchtlich, während die norddeutsche überhaupt nicht zu expandieren schien. Dies ist möglicherweise darauf zurückzuführen, dass die norddeutsche Population jünger als die westdeutsche ist, das Verschleppungsereignis noch nicht so weit zurückliegt und die Population sich noch in der Gründerphase befindet. Die passive weltweite Verschleppung von Stechmücken wird in der Zukunft vermutlich zunehmen, und die Etablierung und Ausbreitung invasiver Spezies, inklusive der Asiatischen Buschmücke und anderer potenzieller Überträger von Krankheitserregern, werden Europa und Deutschland weiterhin vor herausfordernde Probleme stellen. Das Monitoring der Ausbreitung von Populationen und die Durchführung populationsgenetischer Analysen zur Ermittlung von geographischen Ursprüngen sowie von Wanderungs- und Transportrouten werden helfen, weitere Einschleppungs- und Ausbreitungsereignisse nachzuvollziehen und zu unterbinden und sind daher essenzielle Instrumente des Managements von Mückenvektoren.The present work deals with the distribution and population genetics of the invasive Asian bush mosquito Ae. j. japonicus which has been spreading in Germany since 2008 by human-mediated transportation and dispersal. Aedes j. japonicus is a vector of several arboviruses under laboratory conditions, e.g. dengue virus and chikungunya virus. It has also been found infected in the field with Japanese encephalitis virus, West Nile virus and La Crosse virus. Following several independent submissions of Ae. j. japonicus specimens in the framework of the German citizen science project “Mückenatlas”, a population was discovered in West Germany in 2012. The distribution area of this population was far more north than the assumed northern distribution limit of the species in South Germany. The extent of this population was defined by a short-time monitoring to an area of about 2,000 km2. Developmental stages of five locations in West Germany were subjected to population genetic studies in order to study the relatedness of individuals within the population and with other European populations. For this purpose, seven microsatellite loci were examined and compared. In addition, part of the mitochondrial nad4 gene region was sequenced and screened for nucleotide polymorphisms. The results were compared to data previously collected from Swiss, Austrian/Slovenian and Belgian populations. The microsatellite signature of the West German population was quite different from the ones of the other European populations. Further, nad4 haplotypes never described before from Europe were found. Thus, this population must be assumed to derive from an independent introduction event of mosquitoes from overseas. The exact origin – USA or East Asia – could not be identified. In 2013, two more populations of Ae. j. japonicus were detected in Europe: one in North Germany and the other in the Netherlands. The genetic makeup of individuals of these newly detected populations were analysed as described. Additionally, the genetic makeup of a broader range of individuals from Slovenia was examined, as well as that of individuals from Croatia and southern Germany. Results were compared with previous findings and showed a similar microsatellite signature and identical nad4 haplotypes, indicating that the North German population is a subpopulation of the West German one. The low population density and the comparably small distribution area of the North German population also suggest that the split-off took place not long ago. By contrast, the Dutch population appears to originate from a further introduction event of mosquitoes from overseas. In late summer 2015, the most recent German population of Ae. j. japonicus was detected in Upper Bavaria and adjacent Austria. Population genetics showed this population to be closely related to the formerly described Austrian/Slovenian population while being different from all other German populations suggesting it to be a split-off from the Austrian/Slovenian population. The distribution and spread of Ae. j. japonicus in West and North Germany were observed from the time of detection in 2012/2013 until 2015. In this period, the West German population considerably increased its distribution area, while the North German population did not seem to expand at all. The reasons for this are seen in the North German population being younger than the West German one, the separation incident not dating back very far and the population still being in its founder phase. Passive worldwide displacement of mosquitoes will probably increase in the future and establishment and spread of invasive species, including the Asian bush mosquito and other potential vectors of disease agents, will continue to pose challenging problems in Europe and Germany. Monitoring the dispersal of populations and performing population genetic studies to find out geographic origins as well as migration and transportation routes will help reconstructing and preventing further introduction and spread and are therefore essential tools of mosquito vector management

A new focus of <it>Aedes japonicus japonicus</it> (Theobald, 1901) (Diptera, Culicidae) distribution in Western Germany: rapid spread or a further introduction event?

Abstract Background The Asian bush mosquito, Aedes japonicus japonicus, a potential vector of several viruses, was first detected in Germany in 2008 on the Swiss-German border. In the following years, this invasive species apparently succeeded in establishing populations in southern Germany and in spreading northwards. In 2011, its distribution area already covered large areas of the federal state of Baden-Wurttemberg, and its northernmost German collection point was reported to be close to Stuttgart. Several independent submissions to our laboratories of Ae. j. japonicus specimens in July 2012, originating from the same area in the federal state of North Rhine-Westphalia, western Germany, prompted us to carry out an immediate surveillance in this region in the expectation of finding a further distribution focus of Ae. j. japonicus in Germany. Methods After inspecting the places of residence of the collectors of the submitted mosquito specimens, all kinds of water containers in 123 cemeteries in surrounding towns and villages were checked for mosquito developmental stages. These were collected and kept to produce adults for morphological species identification. One specimen per collection site was identified genetically by COI sequence analysis. Results Aedes j. japonicus adults and immature stages were found in 36 towns/villages that were checked (29%) over an area of approximately 2,000 km2 in southern North Rhine-Westphalia and northern Rhineland Palatinate. The species could not be demonstrated further south when monitoring towards the northernmost previous collection sites in southern Germany. It therefore remains to be elucidated whether the species has entered western Germany from the south, from Belgium in the west where it has been demonstrated to occur locally since 2002, or through a new introduction. Conclusions Aedes j. japonicus is obviously much more widely distributed in Germany than previously thought. It appears to be well adapted, to have a strong expansion tendency and to replace indigenous mosquito species. Thus, a further spread is anticipated and elimination seems hardly possible anymore. The vector potency of the species should be reason enough to thoroughly monitor its future development in Germany.</p

Molecular confirmation of the occurrence in Germany of <it>Anopheles daciae</it> (Diptera, Culicidae)

<p>Abstract</p> <p>Background</p> <p><it>Anopheles daciae</it>, a newly described member of the Maculipennis group, was recently reported from western, southern and eastern Europe. Before its recognition, it had commonly been listed under the name of <it>An. messeae</it>, due to its extreme morphological and genetic similarities. As the sibling species of the Maculipennis group are known to differ in their vector competences for malaria parasites and other pathogens, the occurrence of <it>An. daciae</it> in a given region might have an impact on the epidemiology of mosquito-borne diseases. Mosquito collections from different localities in Germany were therefore screened for <it>An. daciae</it>.</p> <p>Methods</p> <p>Adult and immature Maculipennis group mosquitoes were collected between May 2011 and June 2012 at 23 different sites in eight federal states of Germany. A standard PCR assay was used to differentiate the previously known sibling species while the ITS2 rDNA of specimens preliminarily identified as <it>An. messeae</it>/<it>daciae</it> was sequenced and analysed for species-specific nucleotide differences.</p> <p>Results</p> <p>Four hundred and seventy-seven <it>Anopheles</it> specimens were successively identified to Maculipennis group level by morphology and to species level by DNA-based methods. Four species of the Maculipennis group were registered: <it>An. messeae</it> (n = 384), <it>An. maculipennis</it> (n = 82), <it>An. daciae</it> (n = 10) and <it>An. atroparvus</it> (n = 1). <it>Anopheles daciae</it> occurred at four sites in three federal states of Germany, three of the sites being located in north-eastern Germany (federal states of Brandenburg and Saxony) while one collection site was situated in the northern Upper Rhine Valley in the federal state of Hesse, south-western Germany.</p> <p>Conclusions</p> <p>The detection of <it>An. daciae</it> represents the first recognition of this species in Germany where it was found to occur in sympatry with <it>An. messeae</it> and <it>An. maculipennis</it>. As the collection sites were in both north-eastern and south-western parts of Germany, the species is probably even more widely distributed in Germany than demonstrated, albeit apparently with low population densities. Research is needed that confirms the species status of <it>An. daciae</it> and elucidates its vector competence as compared to <it>An. messeae</it> and the other species of the Maculipennis group, in order to optimize management of possible future outbreaks of diseases caused by pathogen transmission through Maculipennis group mosquitoes.</p

Additional file 1: of Newly discovered population of Aedes japonicus japonicus (Diptera: Culicidae) in Upper Bavaria, Germany, and Salzburg, Austria, is closely related to the Austrian/Slovenian bush mosquito population

Pairwise population matrix of Nei’s genetic distances. The lower the values, the closer the genetic relatedness between the respective populations. (XLSX 9 kb

Unexpected patterns of admixture in German populations of Aedes Japonicus Japonicus (Diptera: Culicidae) underscore the importance of human intervention

The mosquito Aedes japonicus japonicus, originally restricted to temperate East Asia, is now widespread in North America and more recently has become established in Europe. To ascertain the putative number of separate introductions to Europe and examine patterns of expansion we analyzed the genetic makeup of Ae. j. japonicus populations from five cemeteries in North Rhine-Westphalia and Rhineland-Palatinate, two western German federal states, as well as of specimens from populations in Belgium, Switzerland, and Austria/Slovenia. To do so, we genotyped individual specimens at seven pre-existing polymorphic microsatellite loci and sequenced part of the nad4 mitochondrial locus. We found evidence of two different genotypic signatures associated with different nad4 mitochondrial haplotypes, indicating at least two genetically differentiated populations of Ae. j. japonicus in Europe (i.e. two distinct genotypes). Belgian, Swiss, and Austrian/Slovenian populations all share the same genotypic signature although they have become differentiated since isolation. Contrary to expectations, the German Ae. j. japonicus are not closely related to those in Belgium which are geographically nearest but are also highly inbred. German populations have a unique genotype but also evidence of mixing between the two genotypes. Also unexpectedly, the populations closest to the center of the German infestation had the highest levels of admixture indicating that separate introductions did not expand and merge but instead their expansion was driven by punctuated human-mediated transport. Critically, the resulting admixed populations have higher genetic diversity and appear invasive as indicated by their increased abundance and recent spread across western Germany