Jenny Christina Hesson

Effective mosquito control relies on correct identification of the target species as well as timing. The floodwater species Aedes sticticus often co-occurs with other mosquito larvae, and early instar larvae can be difficult to identify morphologically. This makes large scale mosquito control operations difficult, as they depend on timely initiation when larvae are still at early instars. This study therefore aimed to develop a quick molecular larval identification method for reliable confirmation of field sites where Ae. sticticus hatching has begun. Importantly, the method needs to be easy to use for staff with minor training in molecular lab techniques and require a minimum of lab equipment. The developed assay can be used on crude samples without DNA extraction using quantitative PCR (qPCR). It has been validated on >100 field-sampled larvae which were also identified to species using sequencing. This further supported the need for molecular identification since several species were found to co-occur in the same habitat as Ae. sticticus.

Floodwater mosquitoes cause extreme nuisance and are the target for mosquito control in several European countries. In Forshaga municipality, western Sweden, high abundance of the floodwater mosquito Aedes sticticus has been reported in past years. This study describes mosquito abundances in Forshaga before and after initiation of mosquito control using VectoBac G^®, a larvicide based on the pre-toxins of Bti, administered by helicopter. Adult mosquitoes were collected with CDC-miniature light traps baited with carbon dioxide, and larval counts were performed in flooded areas. Both EU and Swedish national regulations determine when mosquito control can be used, and in 2019 abundance of adult mosquitoes reached the national requirement to initiate mosquito control the following year. In 2020 and 2021, flooding caused by the spring-flood in river Klarälven and rainfall induced hatching of floodwater mosquito larvae. In both years larval abundance reached the requirement to perform a mosquito control action. Both control actions were successful with >97% decrease in larval abundance and <1,000 adult mosquitoes collected per trap-night. The Bti-applications did not affect the abundance of non-floodwater mosquitoes; thus, it controlled the extreme nuisance without impact on mosquito community as a whole.

Floodwater mosquitoes are a main cause of mosquito nuisance in several European countries, including Sweden. Since 2002, a large-scale mosquito control programme around the river Dalälven in eastern Sweden targets floodwater mosquito species such as Aedes sticticus. The current study was initiated due to citizen complaints about nuisance mosquitoes around Deje village in Forshaga municipality, a region situated at the river Klarälven in western Sweden. Over three years, 2010-2012, mosquito collections with CDC-light traps and morphological species identification were performed to determine the nuisance causing mosquito species and to describe the seasonality, abundance and species composition of mosquitoes in the area. Over the three years of collections at eight trap sites, mosquito abundance increased with a mean of 282 mosquitoes collected per trap-night in 2010, 702 mosquitoes per trap-night in 2011, and 1,367 mosquitoes per trap-night in 2012. The increase in abundance was mainly due to an increase of floodwater mosquitoes, with Ae. sticticus as the main species. Single traps collected over 5,000 floodwater mosquitoes at several occasions in 2011 and 2012. A correlation between river discharge exceeding 300 m³/s and an increase in floodwater mosquito abundance three weeks later when average air temperatures were around 15 °C could be seen. Interestingly, river discharge was the highest in 2010, however mosquito abundance was higher in both 2011 and 2012. Potentially, the increasing number of diapausing eggs available after each season has contributed to the amplification of the Ae. sticticus population in the region.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host–parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito–mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host–parasite–virus relationships.

Vertical transmission (VT) is a phenomenon of vector-borne diseases where a pathogen is transferred from an infected arthropod mother to her offspring. For mosquito-borne flavi- and alphaviruses, VT is commonly viewed as rare; however, both field and experimental studies report on vertical transmission efficiency to a notably varying degree. It is likely that this reflects the different experimental methods used to test vertical transmission efficiency as well as differences between virus–vector combinations. There are very few investigations of the VT of an alphavirus in a Culex vector. Sindbis virus (SINV) is an arthritogenic alphavirus that utilizes Culex species as main vectors both in the summer transmission season and for its persistence over the winter period in northern latitudes. In this study, we investigated the vertical transmission of the SINV in Culex vectors, both in the field and in experimental settings. The detection of SINV RNA in field-collected egg rafts and emerging adults shows that vertical transmission takes place in the field. Experimentally infected females gave rise to adult offspring containing SINV RNA at emergence; however, three to four weeks after emergence none of the offspring contained SINV RNA. This study shows that vertical transmission may be connected to SINV’s ability to persist throughout northern winters and also highlights many aspects of viral replication that need further study.

Infections caused by arthropod-borne RNA viruses are overrepresented among emerging infectious diseases. Effective methods for collecting, storing, and transporting clinical or biological specimens are needed worldwide for disease surveillance. However, many tropical regions where these diseases are endemic lack analytical facilities and possibility of continuous cold chains, which presents challenges from both a biosafety and material preservation perspective. Whatman^® FTA^® Classic Cards may serve as an effective and safe option for transporting hazardous samples at room temperature, particularly for RNA viruses classified as biosafety level (BSL) 2 and 3 pathogens, from sampling sites to laboratories. In this study, we investigated the biosafety and perseverance of representative alpha- and flaviviruses stored on FTA^® cards. To evaluate the virus inactivation capacity of FTA^® cards, we used Sindbis virus (SINV), chikungunya virus (CHIKV), and Japanese encephalitis virus (JEV). We inoculated susceptible cells with dilution series of eluates from viral samples stored on the FTA^® cards and observed for cytopathic effect to evaluate the ability of the cards to inactivate viruses. All tested viruses were inactivated after storage on FTA^® cards. In addition, we quantified viral RNA of JEV, SINV, and tick-borne encephalitis virus (TBEV) stored on FTA^® cards at 4 °C, 25 °C, and 37 °C for 30 days using two reverse transcriptase quantitative PCR assays. Viral RNA of SINV stored on FTA^® cards was not reduced at either 4 °C or 25 °C over a 30-day period, but degraded rapidly at 37 °C. For JEV and TBEV, degradation was observed at all temperatures, with the most rapid degradation occurring at 37 °C. Therefore, the use of FTA^® cards provides a safe and effective workflow for the collection, storage, and analysis of BSL 2- and 3-virus RNA samples, but there is a risk of false negative results if the cards are stored at higher temperatures for long periods of time. Conscious usage of the cards can be useful in disease surveillance and research, especially in tropical areas where transportation and cold chains are problematic.

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes’ ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs’ effects on their host.