Rediscovery of Culex (Neoculex) martinii Medschid, 1930 (Diptera, Culicidae) in Germany.

The culicid Culex martinii is an extremely rare species in Germany, with only one reference in the scientific literature, according to which larvae were found once in 1935 in the eastern part of the country. During regular mosquito sampling activities with BG-Sentinel traps, Cx. martinii was rediscovered in autumn 2017 at one location in the German federal state of Thuringia. Prompted by this finding, the Peus mosquito collection at the Senckenberg Museum Frankfurt was checked for further specimens of this species. Indeed, it contained Cx. martinii specimens from two sites in southern Germany from 1973 in addition to the published finding from 1935 and further specimens from that latter site from 1936 and 1951. Due to its rarity, little is known on the ecology of Cx. martinii and nothing on a possible vector role.

The Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) in Central Germany: Surveillance in its northernmost distribution area.

The invasive Asian tiger mosquito Aedes albopictus has recently been observed in southern Germany for the first time to reproduce and even overwinter north of the Alps. After the accidental capture of adult specimens in Jena, German federal state of Thuringia, in mid-2015, regular inspections brought forth developmental stages until autumn 2015, indicating local reproduction. Surveillance activities implemented in 2016 showed larvae already in early May, suggesting overwintering, and throughout the season until late October, although population densities remained low. Further sporadic specimens found in 2017 argue for establishment. Jena is located in Central Germany, north of all known distribution areas of Ae. albopictus, with the area of the municipality affected by the tiger mosquito characterised by a relatively mild climate. To check the suitability of the local climate for Ae. albopictus, winter temperatures, measured in a cemetery of Jena where larvae had regularly been found in 2015 and 2016, were analysed and compared with two sites of establishment in southern Germany. The conditions were similar at all three locations, suggesting that the Jena population might also be able to survive in the long term. While the municipality authorities have been informed and education of the Jena citizens to avoid producing potential breeding places has started, insecticidal control has not yet been implemented.

Detection of Usutu, Sindbis, and Batai Viruses in Mosquitoes (Diptera: Culicidae) Collected in Germany, 2011⁻2016.

Due to the emergence of non-endemic mosquito vectors and the recent outbreaks of mosquito-borne diseases, mosquito-borne pathogens are considered an increasing risk to public and animal health in Europe. To obtain a status quo regarding mosquito-borne viruses and their vectors in Germany, 97,648 mosquitoes collected from 2011 to 2016 throughout the country were screened for arboviruses. Mosquitoes were identified to species, pooled in groups of up to 50 individuals according to sampling location and date, and screened with different PCR assays for Flavi-, Alpha- and Orthobunyavirus RNA. Two pools tested positive for Usutu virus-RNA, two for Sindbis virus-RNA, and 24 for Batai virus-RNA. The pools consisted of Culex pipiens s.l., Culex modestus, Culex torrentium, Culiseta sp., Aedes vexans, Anopheles daciae, and Anopheles messeae mosquitoes and could be assigned to nine different collection sites, with seven of them located in northeastern Germany. Phylogenetic analyses of the viral RNA sequences showed relationships with strains of the viruses previously demonstrated in Germany. These findings confirm continuing mosquito-borne zoonotic arbovirus circulation even though only a rather small percentage of the screened samples tested positive. With respect to sampling sites and periods, virus circulation seems to be particularly intense in floodplains and after flooding events when mosquitoes develop in excessive numbers and where they have numerous avian hosts available to feed on.

Mosquito species composition and phenology (Diptera, Culicidae) in two German zoological gardens imply different risks of mosquito-borne pathogen transmission.

Due to their large diversity of potential blood hosts, breeding habitats, and resting sites, zoological gardens represent highly interesting places to study mosquito ecology. In order to better assess the risk of mosquito-borne disease-agent transmission in zoos, potential vector species must be known, as well as the communities in which they occur. For this reason, species composition and dynamics were examined in 2016 in two zoological gardens in Germany. Using different methods for mosquito sampling, a total of 2,257 specimens belonging to 20 taxa were collected. Species spectra depended on the collection method but generally differed between the two zoos, while species compositions and relative abundances varied seasonally in both of them. As both sampled zoos were located in the same climatic region and potential breeding sites within the zoos were similar, the differences in mosquito compositions are attributed to immigration of specimens from surrounding landscapes, although the different sizes of the zoos and the different blood host populations available probably also have an impact. Based on the differences in species composition and the various biological characteristics of the species, the risk of certain pathogens to be transmitted must also be expected to differ between the zoos.

Further reports of Anopheles algeriensis Theobald, 1903 (Diptera: Culicidae) in Germany, with evidence of local mass development.

Anopheles algeriensis, a thermophilic mosquito species widely distributed in the Mediterranean, is supposed to be extremely rare and to occur in very low abundances in central and northern Europe. Being one of seven native Anopheles species, it has been reported from Germany a few times only, with all but one report several decades ago. Only in 2013, the endemic persistence of the species was confirmed when two larval specimens were found north of Hamburg. We here report the trapping of An. algeriensis adults at three additional sites in northeastern Germany, with one of them representing two thirds of all mosquitoes collected over two monitored seasons, 2015 and 2017, and a second one with still 12.3% of all specimens caught during the mosquito season 2016. At a third site, one single female was trapped in 2015. Despite considerable efforts, breeding sites could not be identified at the two locations characterised by the high abundances. Anopheles algeriensis has been shown to be vector-competent for Plasmodium parasites and might locally play a role in malaria epidemiology when abundance is high.

The Nuisance Mosquito Anopheles plumbeus (Stephens, 1828) in Germany-A Questionnaire Survey May Help Support Surveillance and Control.

The mosquito species Anopheles plumbeus is an aggressive biter and a potential vector of malaria parasites and West Nile virus. It occurs naturally at low population densities, as its larval development is adapted to the specific water qualities found in tree holes. However, probably owing to environmental changes, it has recently been observed in several European countries to use increasingly often artificial breeding habitats that may lead to mass development and severe annoyance to humans living close by. The perception of mosquito nuisance, however, is very subjective, and breeding habitats are not always known, thus impeding targeted surveillance and control. To relate nuisance by An. plumbeus to specific environmental conditions, a questionnaire survey was carried out addressing persons who had submitted specimens of this particular mosquito species to the German citizen science project "Mueckenatlas", an instrument of passive mosquito surveillance. The questionnaire was intended to find out whether a nuisance situation linked to An. plumbeus had existed, whether mosquito breeding habitats could be identified and whether control measures had been conducted. Despite some efforts, the participants who claimed to suffer from an An. plumbeus nuisance problem had rarely identified the source of the mosquitoes. Once control measures had been performed on abandoned manure pits, however, the nuisance problem disappeared or mosquito abundance was at least significantly reduced. Nevertheless, no significant effect of abandoned manure pits on the probability of an An. plumbeus nuisance could be demonstrated in a multivariate logistic regression model testing various variables. Instead, a significant positive effect of a disused farm nearby was found. The reason is probably that manure pits as the most frequent source of An. plumbeus mass development are often located on disused farms, without most people's knowledge about their existence. Disused farms are therefore appropriate candidates to consider when it comes to public health issues connected to An. plumbeus such as surveillance of mass development and implementation of control measures.

The thermophilic mosquito species Uranotaenia unguiculata Edwards, 1913 (Diptera: Culicidae) moves north in Germany.

Uranotaenia unguiculata is a thermophilic mosquito species frequently occurring in the Mediterranean. Its first detection in the southern German Upper Rhine Valley in 1994 represented its northernmost distribution limit for a long time. During recent mosquito monitoring activities, two specimens of the species were trapped at different localities, about 70 km apart, in northeastern Germany, some 300-km latitude north of previous collection sites. It is not known whether Ur. unguiculata is vector-competent for disease agents although specimens collected in the field were found infected with West Nile virus and Dirofilaria repens. The finding of the species in northern Germany is probably a further example of mosquito species spreading northwards as a consequence of climate warming.

The Citizen Science Project 'Mueckenatlas' Helps Monitor the Distribution and Spread of Invasive Mosquito Species in Germany.

The citizen science project 'Mueckenatlas' (mosquito atlas) was implemented in early 2012 to improve mosquito surveillance in Germany. Citizens are asked to support the spatiotemporal mapping of culicids by submitting mosquito specimens collected in their private surroundings. The Mueckenatlas has developed into an efficient tool for data collection with close to 30,000 mosquitoes submitted by the end of 2015. While the vast majority of submissions included native mosquito species, a small percentage represented invasive species. The discovery of Aedes albopictus (Skuse) (Diptera: Culicidae), Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) and Aedes koreicus (Edwards) (Diptera: Culicidae) specimens via the Mueckenatlas project prompted targeted monitoring activities in the field which produced additional information on the distribution of these species in Germany. Among others, Mueckenatlas submissions led to the detection of three populations of Ae. j. japonicus in West, North and Southeast Germany in 2012, 2013, and 2015, respectively. As demonstrated by on-site monitoring, the origins of Ae. j. japonicus specimens submitted to the Mueckenatlas mirror the distribution areas of the four presently known German populations as found by active field sampling (the fourth population already reported prior to the launch of the Mueckenatlas). The data suggest that a citizen science project such as the Mueckenatlas may aid in detecting changes in the mosquito fauna and can therefore be used to guide the design of more targeted field surveillance activities.

Emerging mosquito species in Germany-a synopsis after 6 years of mosquito monitoring (2011-2016).

Globalisation and climate change are the main drivers of invasion of non-endemic regions by mosquitoes. Mass transportation of people, animals and goods facilitate accidental long-distance displacement while climate warming supports active spread and establishment of thermophilic species. In the framework of a mosquito-monitoring programme, eight non-indigenous culicid species have been registered in Germany since 2011, with four of them being more or less efficient vectors of disease agents and another four now considered established. The eight newly emerged species include Aedes albopictus, Ae. japonicus, Ae. aegypti, Ae. koreicus, Ae. berlandi, Ae. pulcritarsis, Anopheles petragnani and Culiseta longiareolata. We here review recent findings and at the same time present new findings of specimens of non-native mosquito species in Germany.

Activity of Culicoides spp. (Diptera: Ceratopogonidae) inside and outside of livestock stables in late winter and spring.

Culicoides Latreille, 1809 midge species are the putative vectors of Bluetongue virus (BTV) and Schmallenberg virus (SBV) in Europe. To gain a better understanding of the epidemiology of the diseases, basic knowledge about the overwintering of the vectors is needed. Therefore, we investigated culicoid activity in relation to air temperature at livestock stables during late winter and spring season. Ceratopogonids were captured weekly indoors and outdoors on three cattle farms, three horse farms and one sheep farm in the federal state of Brandenburg, Germany between January and May, 2015 by BG-Sentinel UV-light suction traps. First seasonal activity was measured inside a sheep barn and cattle stables in mid-March, suggesting the existence of a preceding vector-free period. The first species at all trapping sites were members of the Obsoletus Complex followed by Culicoides punctatus (Meigen), 1804 and Culicoides pulicaris (Linnaeus), 1758 simultaneously. In total, 160 collections were made, including 3465 Culicoides specimens with 2790 (80.6%) of them being members of the Obsoletus Complex. The remaining 675 individuals belonged to six other culicoid species. 59.8% of all Culicoides were collected indoors, and almost five times as many midges were sampled on cattle farms as on horse farms. Cattle farms harboured seven species while only two species were found on the horse and the sheep farms, respectively. Temperatures, husbandry practises and the presence/quality of potential breeding sites might be responsible for the difference in species and numbers of caught specimens between livestock holdings.

Anopheles plumbeus (Diptera: Culicidae) in Germany: updated geographic distribution and public health impact of a nuisance and vector mosquito.

OBJECTIVES: The aim of this study was to map the current spatial distribution of Anopheles plumbeus in Germany, a potential vector of malaria parasites and West Nile virus. Reports of mass occurrence and nuisance connected with artificial breeding site usage by this species were analysed. METHODS: Distribution data were collected from 2011 to 2014 mainly through trapping and submissions of adult mosquito specimens to a citizen science project. In the framework of the latter, additional information was gathered on recent nuisance incidents caused by An. plumbeus, including a longitudinal analysis of mosquito occurrence and the impact of management measures at a nuisance site in south-western Germany. RESULTS: Based on the most comprehensive set of collection data obtained during the last decades, An. plumbeus is shown to be widely distributed over Germany. The data also indicate a continuing extension of the breeding site repertoire of the species from natural to artificial habitats that facilitate mass development. Increasing incidents of persistent nuisance suggest that this mosquito species is rarely diagnosed correctly and managed adequately. CONCLUSIONS: As An. plumbeus is both a serious nuisance pest and a potential vector species, awareness of this species and the public health problems linked to it should be raised among pest managers and public health personnel.

Occurrence and Spread of the Invasive Asian Bush Mosquito Aedes japonicus japonicus (Diptera: Culicidae) in West and North Germany since Detection in 2012 and 2013, Respectively.

The invasive Asian bush mosquito Aedes japonicus japonicus was first recognised as established in Germany in 2008. In addition to the first known and quickly expanding population in the southwestern part of the country, three separate populations were discovered in West, North and southeastern Germany in 2012, 2013 and 2015, respectively, by means of the 'Mueckenatlas', a German instrument of passive mosquito surveillance. Since the first findings of mosquito specimens in West and North Germany, these regions were checked annually for continuing colonisation and spread of the species. Both affected areas were covered by a virtual 10x10km2 grid pattern in the cells of which cemeteries were screened for immature stages of the mosquito. The cells were considered populated as soon as larvae or pupae were detected, whereas they were classified as negative when no mosquito stages were found in the cemeteries of at least three different towns or villages. Presence was also recorded when Ae. j. japonicus adults were submitted to the 'Mueckenatlas' from the respective cell or when there was evidence of local occurrence in localities other than cemeteries. Based on this approach, a significant expansion of the populated area was documented in West Germany since the first detection of Ae. j. japonicus in 2012 (increase in positive grid cells by more than 400%), while the North German population appears not to be expanding so far (reduction of positive grid cells by ca. 30% since 2013). As Ae. j. japonicus finds suitable climatic and ecological conditions in Germany, the differential expansion of the two populations might be attributed to the West German population being older and thus more firmly established than the closely related but younger North German population that might still be in its founder phase. However, geographic spread of all German populations in the future is anticipated. Continuous surveillance is recommended, as Ae. j. japonicus is a competent vector of several pathogens in the laboratory.

Indoor development of Aedes aegypti in Germany, 2016.

In spring 2016, a German traveller returning from Martinique cultivated imported plant offsets in her home, and accidentally bred Aedes aegypti. Thirteen adult mosquito specimens submitted for identification and the traveller were tested for Zika, dengue and chikungunya virus infections, with negative results. The detection of Ae. aegypti by the 'Mueckenatlas' project demonstrates the value of this passive surveillance scheme for potential public health threats posed by invasive mosquitoes in Germany.

Evidence for an independent third Usutu virus introduction into Germany.

Usutu virus (USUV) is an arbovirus within the genus flavivirus, which was first introduced to Southern Europe approximately twenty years ago causing epizootics among wild and captive birds. In Germany USUV was initially discovered in wild birds, mainly Common blackbirds (Turdus merula), in the Upper Rhine valley in southwest of the country in 2011 and has not spread much northwards since. Phylogenetic analyses revealed that the still ongoing USUV epidemic is caused by two different USUV strains, USUV-Germany belonging to the USUV Europe 3 lineage and USUV-Bonn belonging to the USUV Africa 3 lineage. The two strains were introduced independently. In August 2015 a new USUV strain, named USUV-Berlin, was isolated in Vero cells from two carcasses of juvenile Great grey owls (Strix nebulosa) kept in the Zoological Garden Berlin, which had suffered from a hyperacute fatal systemic infection. Both owls carried high USUV genome loads. Full-length USUV genomes sequences were determined and phylogenetic analysis demonstrated a close relationship with a Spanish mosquito-derived sequence from 2006. Immunohistochemical antigen detection in organ samples of the owls showed the typical USUV infection patterns. According to the phylogenetic analysis, USUV-Berlin belongs to the Africa 2 lineage, and can thus be distinguished from the other strains circulating in Germany. Repeated findings of different USUV strains suggest more frequent introductions into Central Europe and a higher mobility of this virus than assumed to date.

The Anopheles maculipennis complex (Diptera: Culicidae) in Germany: an update following recent monitoring activities.

The Anopheles maculipennis complex comprises several sibling species including major vectors of malaria parasites of historic Europe. In present-day Europe, these species are probably more relevant with regard to transmission of pathogens other than plasmodia, such as viruses and dirofilariae. Distribution data facilitating risk assessments and modelling of An. maculipennis complex-borne diseases, however, are generally outdated. In Germany, the occurrence and geographic distribution of the complex species have recently been updated within the framework of a national monitoring programme. In addition to the known indigenous species An. maculipennis, Anopheles messeae and Anopheles atroparvus, the newly described sibling species Anopheles daciae was demonstrated. Distribution maps of these species based on the data collected from 2011 to 2014 are presented, whilst ecological characteristics and vector roles are presented and discussed.

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.

BACKGROUND: The German mosquito surveillance instrument 'Mueckenatlas' requests the general public to collect and submit mosquito specimens. Among these, increasing numbers of individuals of invasive species have been registered. Specimens of the Asian bush mosquito Aedes japonicus japonicus submitted from German Upper Bavaria, where this species had not previously been recorded, triggered regional monitoring in mid-2015. METHODS: The search for Ae. j. japonicus breeding sites and developmental stages concentrated on cemeteries in the municipality of origin of the submitted specimens and, subsequently, in the whole region. A virtual grid consisting of 10 × 10 km(2) cells in which up to three cemeteries were checked, was laid over the region. A cell was considered positive as soon as Ae. j. japonicus larvae were detected, and regarded negative when no larvae could be found in any of the cemeteries inspected. All cells surrounding a positive cell were screened accordingly. A subset of collected Aedes j. japonicus specimens was subjected to microsatellite and nad4 sequence analyses, and obtained data were compared to individuals from previously discovered European populations. RESULTS: Based on the grid cells, an area of approximately 900 km(2) was populated by Ae. j. japonicus in Upper Bavaria and neighbouring Austria. Genetic analyses of microsatellites and nad4 gene sequences generated one genotype out of two previously described for Europe and three haplotypes, one of which had previously been found in Europe only in Ae. j. japonicus samples from a population in East Austria and Slovenia. The genetic analysis suggests the new population is closely related to the Austrian/Slovenian population. CONCLUSION: As Ae. j. japonicus is well adapted to temperate climates, it has a strong tendency to expand and to colonise new territories in Central Europe, which is facilitated by human-mediated, passive transportation. The new population in Upper Bavaria/Austria is the seventh separate population described in Europe. According to our data, it originated from a previously detected population in eastern Austria/Slovenia and not from an introduction event from abroad. The dispersal and population dynamics of Ae. j. japonicus should be thoroughly surveyed, as this species is a potential vector of disease agents.