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We report an outbreak investigation of two fatal cases of autochthonous Plasmodium falciparum malaria that occurred in Belgium in September 2020. Various hypotheses of the potential source of infection were investigated. The most likely route of transmission was through an infectious exotic Anopheles mosquito that was imported via the international airport of Brussels or the military airport Melsbroek and infected the cases who lived at 5 km from the airports. Based on genomic analysis of the parasites collected from the two cases, the most likely origin of the Plasmodium was Gabon or Cameroon. Further, the parasites collected from the two Belgian patients were identical by descent, which supports the assumption that the two infections originated from the bite of the same mosquito, during interrupted feeding. Although airport malaria remains a rare event, it has significant implications, particularly for the patient, as delayed or missed diagnosis of the cause of illness often results in complications and mortality. Therefore, to prevent such severe or fatal outcomes, we suggest a number of public health actions including increased awareness among health practitioners, especially those working in the vicinity of airports, and increased surveillance of exotic mosquito species at airports.

In May–June 2021, we collected Aedes albopictus (Skuse, 1894) adults near the central hospital in Boende, the capital of Tshuapa province in the Democratic Republic of the Congo (DRC). We identified the mosquitoes using morphological and DNA-based techniques. This is the first report of this species in the DRC outside of Kinshasa and Kongo Central. Given the central location of Boende in the Congo Basin, our finding indicates that the vector might also have spread to other cities which are located on the Congo River and its major tributaries. We highlight the need to investigate the distribution of Ae. albopictus and to update disease risk maps for Central Africa, as it is an important vector for human arbovirus transmission.

In May-June 2021, we detected Aedes albopictus adults near the central hospital in Boende, the capital city of the Tshuapa province of the Democratic Republic of the Congo (DRC). We identified the mosquitoes using morphological and molecular techniques (COI barcoding). This is the first report of this species in the DRC outside of Kinshasa and Kongo Central. Given the central location of Boende in the Congo Basin, our finding suggests that the vector might also have spread to other cities which are located upstream of the Congo River and its major tributaries. Because Aedes albopictus is an important vector for human arbovirus transmission, we highlight the need to investigate its distribution range and to update disease risk maps in Central Africa.

Culiseta (Allotheobaldia) longiareolata (Macquart) (Diptera: Culicidae) is an ornithophilic mosquito species that occurs in the southern Palaearctic Region from the Azores to Central Asia, the Ethiopian Region, India, and Pakistan. Although it has a widespread distribution range, the species was only recently reported in Western and Central Europe. Between 2017 and 2020, larvae, pupae, and adults of Cs. longiareolata (n = 161) were found at 13 distinct locations in Belgium (n = 4) and The Netherlands (n = 9). Collected mosquitoes were morphologically identified and the identification was then validated by COI DNA barcoding. These are the first records of the species in the above-mentioned countries. The present results suggest that Cs. longiareolata could be increasing its distribution range in temperate regions, indicating a warming climate. As the species might be a potential vector of bird pathogens (e.g., West Nile virus), its spread in Western Europe is noteworthy.

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species‐diagnostic PCR‐RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR‐RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co‐occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study., Species identification of 175 specimens of Anopheles maculipennis s.l. collected in Belgium, comparing the performance of COI and ITS2 loci, and two species‐diagnostic PCR‐RFLP assays.Four species identified: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2), with An. daciae reported for the first time in Belgium.Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus (malaria vector). Anopheles daciae and An. messeae appeared the most closely related.

Background Aedes japonicus japonicus has expanded beyond its native range and has established in multiple European countries, including Belgium. In addition to the population located at Natoye, Belgium, locally established since 2002, specimens were recently collected along the Belgian border. The first objective of this study was therefore to investigate the origin of these new introductions, which were assumed to be related to the expansion of the nearby population in western Germany. Also, an intensive elimination campaign was undertaken at Natoye between 2012 and 2015, after which the species was declared to be eradicated. This species was re-detected in 2017, and thus the second objective was to investigate if these specimens resulted from a new introduction event and/or from a few undetected specimens that escaped the elimination campaign. Methods Population genetic variation at nad4 and seven microsatellite loci was surveyed in 224 and 68 specimens collected in Belgium and Germany, respectively. German samples were included as reference to investigate putative introduction source(s). At Natoye, 52 and 135 specimens were collected before and after the elimination campaign, respectively, to investigate temporal changes in the genetic composition and diversity. Results At Natoye, the genotypic microsatellite make-up showed a clear difference before and after the elimination campaign. Also, the population after 2017 displayed an increased allelic richness and number of private alleles, indicative of new introduction(s). However, the Natoye population present before the elimination programme is believed to have survived at low density. At the Belgian border, clustering results suggest a relation with the western German population. Whether the introduction(s) occur via passive human-mediated ground transport or, alternatively, by natural spread cannot be determined yet from the dataset. Conclusion Further introductions within Belgium are expected to occur in the near future, especially along the eastern Belgian border, which is at the front of the invasion of Ae. japonicus towards the west. Our results also point to the complexity of controlling invasive species, since 4 years of intense control measures were found to be not completely successful at eliminating this exotic at Natoye. Graphical Abstract

The Barcoding Facility for Organisms and Tissues of Policy Concern (BopCo) aims at developing an expertise forum to facilitate the identification of biological samples of policy concern in Belgium and Europe. The project represents part of the Belgian federal contribution to the European Research Infrastructure Consortium LifeWatch. Non-native species which are being introduced into Europe, whether by accident or deliberately, can be of policy concern since some of them can reproduce and disperse rapidly in a new territory, establish viable populations and even outcompete native species. As a consequence of their presence, natural and managed ecosystems can be disrupted, crops and livestock affected, and vector-borne diseases or parasites might be introduced, impacting human health and socio-economic activities. Non-native species causing such adverse effects are called Invasive Alien Species (IAS). In order to protect native biodiversity and ecosystems, and to mitigate the potential impact on human health and socio-economic activities, the issue of IAS is tackled in Europe by EU Regulation 1143/2014 of the European Parliament and Council. The IAS Regulation provides for a set of measures to be taken across all member states. The list of Invasive Alien Species of Union Concern is regularly updated. In order to implement the proposed actions, however, methods for accurate species identification are required when suspicious biological material is encountered. Because morphology-based species identifications are not always possible (e.g. cryptic species, trace material, early life-stages), the purpose of the present work is to investigate and evaluate the usefulness of DNA sequence data to identify each of the IAS included in the EU Regulation. The results are presented as factsheets (one per IAS) compiled using publicly available DNA sequence data and information aggregated from various sources. Each factsheet consists of two major parts; (i) a short introduction to the specific IAS compiling information on its taxonomy and current occurrence/distribution in Europe; (ii) an investigation with respect to the usefulness of publicly available DNA sequences to identify this IAS to the taxonomic level stated in the EU list using DNA barcoding. For further information about the reasoning behind the applied approach and details on the materials and methods utilised, please see below and Smitz et al. [1].&nbsp