First record of the invasive mosquito species Aedes koreicus (Diptera: Culicidae) in the Czech Republic.

Aedes koreicus is an invasive mosquito species originating from East Asia. It has recently been introduced into several countries in Southern, Central and Eastern Europe as well as Central Asia in many of which it has successfully established populations. The biology and ecological requirements of the species are largely unknown, but it is considered as a potential vector of pathogens that requires careful monitoring. We report here the first detection of Ae. koreicus in the Czech Republic, based on a citizen report.

Emergence of the invasive Asian bush mosquito Aedes (Hulecoeteomyia) japonicus (Theobald, 1901) in the Czech Republic.

BACKGROUND: Aedes japonicus is a mosquito species native to North-East Asia that was first found established outside its original geographic distribution range in 1998 and has since spread massively through North America and Europe. In the Czech Republic, the species was not reported before 2021. METHODS: Aedes invasive mosquitoes (AIM) are routinely surveyed in the Czech Republic by ovitrapping at potential entry ports. This surveillance is supported by appeals to the population to report uncommon mosquitoes. The submission of an Ae. japonicus specimen by a citizen in 2021 was followed by local search for aquatic mosquito stages in the submitter's garden and short-term adult monitoring with encephalitis virus surveillance (EVS) traps in its surroundings. Collected Ae. japonicus specimens were subjected to nad4 haplotype and microsatellite analyses. RESULTS: Aedes japonicus was detected for the first time in the Czech Republic in 2021. Aquatic stages and adults were collected in Prachatice, close to the Czech-German border, and eggs in Mikulov, on the Czech-Austrian border. Morphological identification was confirmed by molecular taxonomy. Genetic analysis of specimens and comparison of genetic data with those of other European populations, particularly from Germany, showed the Prachatice specimens to be most closely related to a German population. The Mikulov specimens were more distantly related to those, with no close relatives identifiable. CONCLUSIONS: Aedes japonicus is already widely distributed in Germany and Austria, two countries neighbouring the Czech Republic, and continues to spread rapidly in Central Europe. It must therefore be assumed that the species is already present at more than the two described localities in the Czech Republic and will further spread in this country. These findings highlight the need for more comprehensive AIM surveillance in the Czech Republic.

First Autochthonous West Nile Lineage 2 and Usutu Virus Infections in Humans, July to October 2018, Czech Republic.

We present epidemiological, clinical and laboratory findings of five Czech patients diagnosed with autochthonous mosquito-borne disease-four patients with confirmed West Nile virus (WNV) and one patient with Usutu virus (USUV) infections, from July to October 2018, including one fatal case due to WNV. This is the first documented human outbreak caused by WNV lineage 2 in the Czech Republic and the first record of a neuroinvasive human disease caused by USUV, which illustrates the simultaneous circulation of WNV and USUV in the country.

First Record of Mosquito-Borne Kyzylagach Virus in Central Europe.

RNA of Kyzylagach virus (KYZV), a Sindbis-like mosquito-borne alphavirus from Western equine encephalitis virus complex, was detected in four pools (out of 221 pools examined), encompassing 10,784 female Culex modestus mosquitoes collected at a fishpond in south Moravia, Czech Republic, with a minimum infection rate of 0.04%. This alphavirus was never detected in Central Europe before.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Overwintering of Culex modestus and Other Mosquito Species in a Reedbed Ecosystem, Including Arbovirus Findings.

The overwintering strategy of the mosquito Culex modestus, an important West Nile virus (WNV) vector in Europe, was explored under field conditions in reedbed (Phragmites australis) ecosystems in early 2019. A total of 30 Cx. modestus females were found in a BG-Sentinel trap placed in a plastic greenhouse as well as in a reference BG-Sentinel trap placed under the open sky, both set up within the reedbeds and inspected every 2-3 days from February 27 to April 10, 2019. Moreover, 186 females of Cx. pipiens, 3 females of Anopheles hyrcanus, and 3 females of Culiseta annulata were trapped in the monitored time span. While all Cx. modestus females tested negative for the presence of WNV and other arboviruses circulating in Central Europe, we confirmed WNV lineage 2 and Tahyna virus infection in several pools of the collected Cx. pipiens, demonstrating arbovirus overwintering. This pilot study highlights the need for large-scale monitoring activities covering different regions to identify the overwintering strategy of both mosquito-borne viruses and their vectors in Central Europe.

Co-Circulation of West Nile and Usutu Flaviviruses in Mosquitoes in Slovakia, 2018.

Monitoring West Nile virus (WNV) and Usutu virus (USUV) activity now has the highest priority among mosquito-borne pathogenic viruses circulating in the European Union. This study documents a first time detection and the co-circulation of WNV lineage-2 (with the minimal prevalence of 0.46%) and USUV clade Europe 2 (with the minimal prevalence of 0.25%) in mosquitoes from the same habitat of south-western Slovakia and underlines necessity to perform rigorous surveillance in birds, mosquitoes, horses and humans in that country.

The invasive Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) in the Czech Republic: Repetitive introduction events highlight the need for extended entomological surveillance.

In the framework of a regional collaborative project between authorities and scientists, evidence was found of repeated introduction of Aedes (Stegomyia) albopictus (Skuse) alongside the main road entrances (E461 and E65) connecting Austria and the Slovak Republic with the Czech Republic. In comparison to data from 2012 (17 specimens collected on three occasions), the seasons 2016 (66 specimens on ten occasions) and 2017 (90 specimens on eight occasions) show an apparent increase of introduction events as well as of mosquito numbers and underline the need for more intense surveillance activities.

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.

West Nile virus in overwintering mosquitoes, central Europe.

BACKGROUND: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far. RESULTS: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites. CONCLUSIONS: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.

Overwintering of Uranotaenia Unguiculata Adult Females in Central Europe: A Possible Way of Persistence of the Putative New Lineage of West Nile Virus?

We report the overwintering of Uranotaenia unguiculata adult females in Central Europe (Czech Republic, Hungary, Austria). This finding suggests a potential mode of winter persistence of putative novel lineage of West Nile virus in the temperate regions of Europe.

Co-circulation of Usutu virus and West Nile virus in a reed bed ecosystem.

BACKGROUND: Mosquito-borne flaviviruses are a major public health threat in many countries worldwide. In Central Europe, West Nile virus (WNV) and Usutu virus (USUV), both belonging to the Japanese encephalitis virus group (Flaviviridae) have emerged in the last decennium. Surveillance of mosquito vectors for arboviruses is a sensitive tool to evaluate virus circulation and consequently to estimate the public health risk. METHODS: Mosquitoes (Culicidae) were collected at South-Moravian (Czech Republic) fishponds between 2010 and 2014. A total of 61,770 female Culex modestus Ficalbi mosquitoes, pooled to 1,243 samples, were examined for flaviviruses by RT-PCR. RESULTS: One pool proved positive for USUV RNA. Phylogenetic analysis demonstrated that this Czech USUV strain is closely related to Austrian and other Central European strains of the virus. In addition, nine strains of WNV lineage 2 were detected in Cx. modestus collected in the same reed bed ecosystem. CONCLUSIONS: This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses.

Zoonotic Dirofilaria repens (Nematoda: Filarioidea) in Aedes vexans mosquitoes, Czech Republic.

The surveillance of vectors for arthropod-borne pathogens is nowadays an important tool in surveillance programmes throughout Europe. Whereas many studies have been performed to screen arthropods for viruses or bacterial pathogens, only limited information is available concerning the geographical distribution and vector range of pathogenic filariae in Central Europe. To consider the prevalence of filarial parasites in mosquito vectors, we performed a molecular survey of mosquitoes for filarial DNA. Mosquito collection was conducted at six study sites in the South Moravian region (Czech Republic) close to the borders with Slovakia and Austria from 2009 to 2011. Molecular screening of mosquitoes was conducted using conventional PCR with primers designed to amplify the mitochondrial cytochromoxidase subunit I gene as well as the partial 5.8S ribosomal RNA gene. A total of 13,222 mosquitoes belonging to six species were captured and distributed into 237 pools with different numbers of individuals. Overall, four pools were positive for Dirofilaria repens (a minimum infection rate 0.03%) at two study sites (both natural and urban). Another filarial parasite detected during a study into Aedes vexans mosquitoes revealed the closest homology to Setaria spp. We detected specific D. repens DNA in Ae. vexans mosquitoes for the first time in the Czech Republic and confirmed the circulation of Dirofilaria spp. in a natural focus of infection providing an epidemiological link between autochthonous canine cases and mosquito vectors in the area studied.

Worthy of their name: how floods drive outbreaks of two major floodwater mosquitoes (Diptera: Culicidae).

An understanding of how climate variables drive seasonal dynamics of mosquito populations is critical to mitigating negative impacts of potential outbreaks, including both nuisance effects and risk of mosquito-borne infectious disease. Here, we identify climate variables most affecting seasonal dynamics of two major floodwater mosquitoes, Aedes vexans (Meigen, 1830) and Aedes sticticus (Meigen, 1838) (Diptera: Culicidae), along the lower courses of the Dyje River, at the border between the Czech Republic and Austria. Monthly trap counts of both floodwater mosquitoes varied both across sites and years. Despite this variability, both models used to fit the observed data at all sites (and especially that for Ae. sticticus) and site-specific models fitted the observed data quite well. The most important climate variables we identified-temperature and especially flooding-were driving seasonal dynamics of both Aedes species. We suggest that flooding determines seasonal peaks in the monthly mosquito trap counts while temperature modulates seasonality in these counts. Hence, floodwater mosquitoes indeed appear worthy of their name. Moreover, the climate variables we considered for modeling were able reasonably to predict mosquito trap counts in the month ahead. Our study can help in planning flood management; timely notification of people, given that these mosquitoes are a real nuisance in this region; public health policy management to mitigate risk from such mosquito-borne diseases as that caused in humans by the Tahyna virus; and anticipating negative consequences of climate change, which are expected only to worsen unless floods, or the mosquitoes themselves, are satisfactorily managed.

Detection of mosquito-only flaviviruses in Europe.

The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy, Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.