Background: The common house mosquito Culex pipiens is known to be a major vector for West Nile virus. In order to decrease risks of West Nile virus outbreaks in Europe, insecticides and the bio-larvicide Bacillus thuringiensis israelensis (Bti) are commonly used for vector control. Alarmingly, insecticide resistance has been reported in Cx. pipiens populations from Southern Europe and several countries neighbouring Europe. For Central and Northern Europe, however, the phenotypic insecticide resistance status of Cx. pipiens has not yet been investigated. Methods: A literature review was performed to assess the geographical distribution of insecticide resistance in Cx. pipiens. To fill the gap of knowledge for Central and Northern Europe, WHO susceptibility tests with permethrin, deltamethrin, malathion, bendiocarb and DDT and a larval toxicity test with Bti were performed with a Cx. pipiens population from Belgium, a country in Central Europe. Results: This research provides the first evidence of widespread phenotypic insecticide resistance in Cx. pipiens. In general, Cx. pipiens developed resistance against multiple insecticides in several countries. Another Cx. pipiens population from Belgium was tested and showed insecticide resistance against deltamethrin, permethrin, DDT and possibly against bendiocarb. The bio-larvicide Bti caused lower mortality than reported for other Cx. pipiens populations in the literature. Conclusions: These results indicate the urgent need for insecticide resistance monitoring against commonly used adulticides and larvicides in Europe, for the translation of knowledge gained regarding the limited efficiency and availability of insecticide into EU legislation and the need for innovative non-chemical vector control tools in order to counter the widespread insecticide resistance in Culex populations. Graphical

Invasive mosquito species (IMS) and their associated mosquito-borne diseases are emerging in Europe. In Belgium, the first detection of Aedes albopictus (Skuse 1894) occurred in 2000 and of Aedes japonicus japonicus (Theobald 1901) in 2002. Early detection and control of these IMS at points of entry (PoEs) are of paramount importance to slow down any possible establishment. This article reviews the introductions and establishments recorded of three IMS in Belgium based on published (2007–2014) and unpublished (2015–2020) data collected during several surveillance projects. In total, 52 PoEs were monitored at least once for the presence of IMS between 2007 and 2020. These included used tyre and lucky bamboo import companies, airports, ports, parking lots along highways, shelters for imported cutting plants, wholesale markets, industrial areas, recycling areas, cemeteries and an allotment garden at the country border with colonised areas. In general, monitoring was performed between April and November. Mosquitoes were captured with adult and oviposition traps as well as by larval sampling. Aedes albopictus was detected at ten PoEs, Ae. japonicus at three PoEs and Aedes koreicus (Edwards 1917) at two PoEs. The latter two species have established overwintering populations. The percentage of PoEs positive for Ae. albopictus increased significantly over years. Aedes albopictus is currently entering Belgium through lucky bamboo and used tyre trade and passive ground transport, while Ae. japonicus through used tyre trade and probably passive ground transport. In Belgium, the import through passive ground transport was first recorded in 2018 and its importance seems to be growing. Belgium is currently at the invasion front of Ae. albopictus and Ae. japonicus. The surveillance and control management actions at well-known PoEs associated to long-distance introductions are more straightforward than at less-defined PoEs associated with short-distance introductions from colonised areas. These latter PoEs represent a new challenge for IMS management in Belgium in the coming years. Aedes albopictus is expected to become established in Belgium in the coming years, hence increasing the likelihood of local arbovirus transmission. The implementation of a sustainable, structured and long-term IMS management programme, integrating active and passive entomological surveillance, vector control and Public Health surveillance is therefore pivotal. Graphical Abstract

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.

This survey reports on the DNA identification and occurrence of Culex torrentium and Cx. pipiens s.s. in Belgium. These native disease-vector mosquito species are morphologically difficult to separate, and the biotypes of Cx. pipiens s.s. are morphologically indistinguishable. Culex torrentium and Cx. pipiens s.s. were identified using the COI and ACE2 loci. We recorded 1248 Cx. pipiens s.s. and 401 Cx. torrentium specimens from 24 locations in Belgium (collected between 2017 and 2019). Culex pipiens biotypes pipiens and molestus, and their hybrids, were differentiated using fragment-size analysis of the CQ11 locus (956 pipiens and 227 molestus biotype specimens, 29 hybrids). Hybrids were observed at 13 out of 16 sympatric sites. These results confirm that both species are widespread in Belgium, but while Cx. torrentium revealed many COI haplotypes, Cx. pipiens s.s. showed only one abundant haplotype. This latter observation may either reflect a recent population-wide demographic or range expansion, or a recent bottleneck, possibly linked to a Wolbachia infection. Finally, new evidence is provided for the asymmetric but limited introgression of the molestus biotype into the pipiens biotype.

The invasive mosquito species Aedes albopictus (Skuse, 1894) is rapidly spreading in Europe, posing an increasing threat because of its high vector competence for chikungunya and dengue virus. An integrative and eco-friendly control of these populations is required to prevent mosquito-borne disease outbreaks. Traditionally-used insecticides or other chemical control agents are often expensive, harmful to the environment, strictly controlled or completely banned in several countries. Additionally, insecticide resistance is a potential threat. One possibility for biological control agents is the use of native aquatic beetles as natural predators of mosquitoes to boost Bacillus thuringiensis israelensis (Bti) interventions. Thirty predatory aquatic beetle taxa were caught in Belgium and kept at the Institute of Tropical Medicine’s insectary to test predation rate and prey choice on Aedes albopictus and Culex pipiens Linnaeus, 1758. Predation rates suggest at least four efficient dytiscid predators that are known to inhabit small, temporary habitats in Europe. Further experiments on prey choice reveal a clear preference for Aedes albopictus over alternative larval prey (Culex pipiens, Daphnia sp., Chaoboridae). We found a strong ecological overlap of the feeding niche of A. albopictus and the hunting zone of dytiscid predators in the benthic layer of small waterbodies. Our findings on the efficacy are very encouraging to further assess the potential of native predacious diving beetles as a biological control agent against the invasive A. albopictus in Europe.

Sand flies are vectors of Leishmania spp. protozoa, phleboviruses and Bartonella bacilliformis. In this study we surveyed areas of central-western Europe, encompassing the northern limit of the known sand fly distribution, and investigated the relationship between their presence and environmental variables. In this area, very limited occurrence data exists, none of them recent. Sampling was performed in July and August 2023 using CDC-light traps, sticky traps and human landing captures, at 179 selected sites in 48 municipalities. A total of 55 sand fly specimens were collected at 11% (20/179) of the sites sampled – including 16 sites in France, 2 in Luxembourg and 1 in Germany – comprising the first published records for Luxembourg and the Trier-Saarburg County of Germany. No sand flies were detected in Belgium or in the Netherlands. Two species were captured: Phlebotomus mascittii (37 females, 2 males) and Ph. perniciosus (1 female, 15 males). The latter species was only found in Savoie, the southernmost region sampled in France, while the former species was detected as far north as latitude 50°N. Logistic regression modelling indicated that the probability of sand fly presence gradually decreased with increasing latitude and altitude (P < 0.05), and it was not associated to other analysed landscape features in the proximity of the traps. The study confirms and provides new evidence for the presence of sand flies in areas of Western Europe and highlights the need to be aware of potential autochthonous transmission of sand fly-borne pathogens in these areas.