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.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

West Nile Virus in Slovenia.

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient's urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.

Predicting the success of an invader: Niche shift versus niche conservatism.

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype-environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat-shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental-induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.

Presence and Potential Distribution of Aedes albopictus and Aedes japonicus japonicus (Diptera: Culicidae) in Slovenia.

In Slovenia, two invasive mosquito species are present, Aedes albopictus (Skuse, 1895) (Diptera: Culicidae) and Aedes japonicus (Theobald, 1901) (Diptera: Culicidae). In this study, we examined their actual distribution and suitable habitats for new colonizations. Data from survey of species presence in 2013 and 2015, bioclimatic variables and altitude were used for the construction of predictive maps. We produced various models in Maxent software and tested two bioclimatic variable sets, WorldClim and CHELSA. For the variable selection of A. albopictus modeling we used statistical and expert knowledge-based approach, whereas for A. j. japonicus we used only a statistically based approach. The best performing models for both species were chosen according to AIC score-based evaluation. In 2 yr of sampling, A. albopictus was largely confined to the western half of Slovenia, whereas A. j. japonicus spread significantly and can be considered as an established species in a large part of the country. Comparison of models with WorldClim and CHELSA variables for both species showed models with CHELSA variables as a better tool for prediction. Finally, we validated the models performance in predicting distribution of species according to collected field data. Our study confirms that both species are co-occurring and are sympatric in a large part of the country area. The tested models could be used for future prevention of invasive mosquitoes spreading in other countries with similar bioclimatic conditions.

First record of Aedes koreicus (Diptera, Culicidae) in Slovenia.

The first record of Aedes koreicus was made in the village of Lovrenc na Dravskem Polju, north-eastern part of the country. The discovery of Ae. koreicus in various continental European countries motivated us to revise samples of the collected Aedes japonicus japonicus. We found Ae. koreicus in samples from 2013, where the larvae were misidentified as Ae. j. japonicus. The species was identified morphologically and molecularly. The first discovery of Ae. koreicus advocates an urgent need for a nationwide mosquito surveillance programme.

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.