Rody Blom

The two biotypes of the mosquito species Culex pipiens s.s. (biotype pipiens and biotype molestus) differ in overwintering strategy. Biotype pipiens enters diapause, whereas biotype molestus remains active year-round. In the Netherlands, flooded crawlspaces may provide sheltered development sites for biotype molestus in winter. We therefore investigated the effects of human population density and crawlspace flooding on the presence of Cx. pipiens s.s. biotypes and their hybrids using citizen-submitted mosquito samples via the platform ‘Muggenradar.’ Mosquitoes were categorized by population density and crawlspace condition. In addition, biotypes and feeding patterns were identified using molecular analyses. Descriptive analyses indicated that biotype molestus was more abundant in areas with high population density (24.1% at ’low’ versus 58.6% at ‘high’), while biotype pipiens dominated in areas of low population density (19.5% at ‘high’ versus 63.3% at ‘low’). Similarly, we found an association of biotype molestus with flooded crawlspaces (68.4% of specimens). However, these patterns were not fully supported by a multivariate analysis. Blood meal analyses revealed that nearly all biotype molestus (98.2%) and hybrid mosquitoes (100%) fed on humans. Surprisingly, six blood-engorged biotype pipiens were collected. We conclude that anthropogenic environments may influence the occurrence of Cx. pipiens s.s. biotypes and hybrids in winter.

Background Culex pipiens sensu stricto (s.s.) is considered the primary vector of Usutu virus and West Nile virus, and consists of two morphologically identical but behaviourally distinct biotypes (Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus) and their hybrids. Both biotypes are expected to differ in their feeding behaviour, and pipiens/molestus hybrids are presumed to display intermediate feeding behaviour. However, the evidence for distinct feeding patterns is scarce, and to date no studies have related differences in feeding patterns to differences in host abundance. Methods Mosquitoes were collected using CO2-baited traps. We collected blood-engorged Cx. pipiens/torrentium specimens from 12 contrasting urban sites, namely six city parks and six residential areas. Blood engorged Cx. pipiens/torrentium mosquitoes were identified to the species and biotype/hybrid level via real-time polymerase chain reaction (PCR). We performed blood meal analysis via PCR and Sanger sequencing. Additionally, avian host communities were surveyed via vocal sounds and/or visual observation. Results We selected 64 blood-engorged Cx. pipiens/torrentium mosquitoes of which we successfully determined the host origin of 55 specimens. Of these, 38 belonged to biotype pipiens, 14 were pipiens/molestus hybrids and the identity of three specimens could not be determined. No blood-engorged biotype molestus or Cx. torrentium specimens were collected. We observed no differences in feeding patterns between biotype pipiens and pipiens/molestus hybrids across different habitats. Avian community composition differed between city parks and residential areas, whereas overall avian abundance did not differ between the two habitat types. Conclusions Our results show the following: (1) Cx. pipiens s.s. feeding patterns did not differ between city parks and residential areas, regardless of whether individuals were identified as biotype pipiens or pipiens/molestus hybrids. (2) We detected differences in host availability between city parks and residential areas. (3) We show that in both urban habitat types, biotype pipiens and pipiens/molestus hybrids fed on both mammalian and avian hosts. This underscores the potential role in arbovirus transmission of biotype pipiens and pipiens/molestus hybrids.

On 22 August, a common whitethroat in the Netherlands tested positive for West Nile virus lineage 2. The same bird had tested negative in spring. Subsequent testing of Culex mosquitoes collected in August and early September in the same location generated two of 44 positive mosquito pools, providing first evidence for enzootic transmission in the Netherlands. Sequences generated from the positive mosquito pools clustered with sequences that originate from Germany, Austria and the Czech Republic.

The incidence and risk of mosquito-borne disease outbreaks in Northwestern Europe has increased over the last few decades. Understanding the underlying environmental drivers of mosquito population dynamics helps to adequately assess mosquito-borne disease risk. While previous studies have focussed primarily on the effects of climatic conditions (i.e., temperature and precipitation) and/or local environmental conditions individually, it remains unclear how climatic conditions interact with local environmental factors such as land use and soil type, and how these subsequently affect mosquito abundance. Here, we set out to study the interactive effects of land use, soil type and climatic conditions on the abundance of Culex pipiens/torrentium, highly abundant vectors of West Nile virus and Usutu virus. Mosquitoes were sampled at 14 sites throughout the Netherlands. At each site, weekly mosquito collections were carried out between early July and mid-October 2020 and 2021. To assess the effect of the aforementioned environmental factors, we performed a series of generalized linear mixed models and non-parametric statistical tests. Our results show that mosquito abundance and species richness consistently differ among land use- and soil types, with peri-urban areas with peat/clay soils having the highest Cx. pipiens/torrentium abundance and sandy rural areas having the lowest. Furthermore, we observed differences in precipitation-mediated effects on Cx. pipiens/torrentium abundance between (peri-)urban and other land uses and soil types. In contrast, effects of temperature on Cx. pipiens/torrentium abundance remain similar between different land use and soil types. Our study highlights the importance of both land use and soil type in conjunction with climatic conditions for understanding mosquito abundances. Particularly in relation to rainfall events, land use and soil type has a marked effect on mosquito abundance. These findings underscore the importance of local environmental parameters for studies focusing on predicting or mitigating disease risk.

Analyses of mosquito-borne virus outbreaks have revealed the presence of similar virus strains over several years. However, it remains unclear how mosquito-borne viruses can persist over winter, when conditions are generally unfavorable for virus circulation. One potential route for virus persistence is via diapausing mosquitoes. We therefore studied whether Usutu virus (USUV), West Nile virus (WNV) and/or Sindbis virus (SINV) can be identified in diapausing mosquitoes in The Netherlands. Mosquito collections were carried out in November 2022 in hibernacula located in two areas with previously observed WNV and/or USUV activity. A total of 4857 mosquitoes, belonging to four species (groups) (Culex pipiens/torrentium, Culiseta annulata, Anopheles maculipennis s.l. and Culex territans), were collected. WNV-, USUV- and SINV-screening using a multiplex real-time RT-PCR assay was carried out on mono-specific mosquito pools. One Culex pipiens/torrentium pool tested positive for USUV RNA. Whole genome sequencing and subsequent phylogenetic analysis revealed that the virus belongs to USUV lineage Africa 3 and clusters with other USUV sequences derived from The Netherlands in 2022. This finding confirms our hypothesis of the potential of local overwintering of USUV in diapausing mosquitoes in The Netherlands.