Wolbachia strains wMel and wAlbB differentially affect Aedes aegypti traits related to fecundity.

Two Wolbachia strains, wMel and wAlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains wMel and wAlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the wMel strain exhibited several significant differences compared with those infected with the wAlbB strain. Specifically, wMel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia-based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia-infected Aedes aegypti: wMel and wAlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that wMel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on mosquito fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations.

Using geometric wing morphometrics to distinguish Aedes japonicus japonicus and Aedes koreicus.

BACKGROUND: Aedes japonicus japonicus (Theobald, 1901) and Aedes koreicus (Edwards, 1917) have rapidly spread in Europe over the last decades. Both species are very closely related and occur in sympatry. Females and males are difficult to distinguish. However, the accurate species discrimination is important as both species may differ in their vectorial capacity and spreading behaviour. In this study, we assessed the potential of geometric wing morphometrics as alternative to distinguish the two species. METHODS: A total of 147 Ae. j. japonicus specimens (77 females and 70 males) and 124 Ae. koreicus specimens (67 females and 57 males) were collected in southwest Germany. The left wing of each specimen was removed, mounted and photographed. The coordinates of 18 landmarks on the vein crosses were digitalised by a single observer. The resulting two-dimensional dataset was used to analyse the differences in the wing size (i.e. centroid size) and wing shape between Ae. j. japonicus and Ae. koreicus using geometric morphometrics. To analyse the reproducibility of the analysis, the landmark collection was repeated for 20 specimens per sex and species by two additional observers. RESULTS: The wing size in female Ae. koreicus was significantly greater than in Ae. j. japonicus but did not differ significantly for males. However, the strong overlap in wing size also for the females would not allow to discriminate the two species. In contrast, the wing shape clustering was species specific and a leave-one-out validation resulted in a reclassification accuracy of 96.5% for the females and 91.3% for the males. The data collected by different observers resulted in a similar accuracy, indicating a low observer bias for the landmark collection. CONCLUSIONS: Geometric wing morphometrics provide a reliable and robust tool to distinguish female and male specimens of Ae. j. japonicus and Ae. koreicus.

First record of Anopheles (Anopheles) hyrcanus (Pallas 1771) (Diptera: Culicidae) in Poland.

The spatial distribution of mosquito species in the course of globalization and climate warming is highly dynamic. Different studies have demonstrated the spread and establishment of thermophilic mosquito species, potentially increasing the prevalence of 'nuisance' mosquitoes and the local transmission of pathogens. Here we report the first recorded sampling of Anopheles hyrcanus in Wroclaw, southwest Poland. This is the most northern detection of this species to date in Europe. Future spread and population development of this potential vector of malaria parasites, viruses or zoonotic helminths, such as Dirofilaria spp., must be monitored carefully. Potential factors underlying the spread of this species are discussed.

Vector Competence of Northern European Culex pipiens Biotype pipiens and Culex torrentium to West Nile Virus and Sindbis Virus.

The West Nile Virus (WNV) and Sindbis virus (SINV) are avian-hosted mosquito-borne zoonotic viruses that co-circulate in some geographical areas and share vector species such as Culex pipiens and Culex torrentium. These are widespread in Europe, including northern parts and Finland, where SINV is endemic, but WNV is currently not. As WNV is spreading northwards in Europe, we wanted to assess the experimental vector competence of Finnish Culex pipiens and Culex torrentium mosquitoes to WNV and SINV in different temperature profiles. Both mosquito species were found susceptible to both viruses and got infected via infectious blood meal at a mean temperature of 18 °C. WNV-positive saliva was detected at a mean temperature of 24 °C, whereas SINV-positive saliva was detected already at a mean temperature of 18 °C. Cx. torrentium was found to be a more efficient vector for WNV and SINV over Cx. pipiens. Overall, the results were in line with the previous studies performed with more southern vector populations. The current climate does not seem optimal for WNV circulation in Finland, but temporary summertime transmission could occur in the future if all other essential factors are in place. More field data would be needed for monitoring and understanding the northward spreading of WNV in Europe.

Honey-baited FTA cards in box gravid traps for the assessment of Usutu virus circulation in mosquito populations in Germany.

Usutu virus (USUV) is becoming increasingly important to veterinary and human health in Germany. USUV has been implicated in mass die-off events of birds, especially of blackbirds (Turdus merula), and has experienced significant range expansion in the years since its first detection in 2010. Current detection methods rely primarily on dead bird surveillance or mass mosquito collection using CO2 as the main attractant. Dead bird surveillance can result in detection of disease circulation past the point at which control efforts would be most impactful. Vector surveillance offers the opportunity to detect disease circulation before significant outbreaks occur. However, current methods result in collections of extremely large numbers of predominantly nulliparous female mosquitoes who have not yet taken a blood meal. This study sought to test whether box gravid traps could successfully trap USUV infected gravid Culex mosquitoes, and if viral RNA could be successfully transferred and stabilised on an FTA card. During the month of August 2020, 18 Reiter-Cummings style box gravid traps with honey-baited FTA cards were set in a region of known USUV circulation around the southern border of Hesse, Germany. Four 48-hour trapping rounds were conducted. All mosquitoes and FTA cards were collected and stored during transport to the laboratory on dry ice. Samples and FTA cards were then transferred and stored in a freezer at -5 °C until identification. Identification was carried out on a chill plate before being sent with overnight courier in a styrofoam box with cooling elements for virus detection with a modified generic flavivirus RT-PCR. Mosquitoes were separated into pools by trap, date, species and feeding status. 2003 mosquitoes were caught in four rounds of trapping, 1834 or 88% of which were female Culex mosquitoes used for examination. 13 pools of mosquitoes and four FTA cards tested positive for USUV. No positive FTA cards were found in traps with positive mosquitoes and no positive mosquitoes were found in traps with positive FTA cards. Although fewer FTA cards than expected returned a positive result, this may have been a result of the extreme conditions experienced in the field and highlights the need to establish the temperature and humidity boundaries such a collection method can withstand. Box gravid traps however, provided a highly effective and targeted approach for capturing gravid female Culex mosquitoes, the most appropriate subpopulation for testing for USUV. Additionally, the simplicity and effectiveness of this trapping and surveillance method make it an attractive option for use as an early warning system, including for large scale surveillance programmes.

Vector Competence of the Invasive Mosquito Species Aedes koreicus for Arboviruses and Interference with a Novel Insect Specific Virus.

The global spread of invasive mosquito species increases arbovirus infections. In addition to the invasive species Aedes albopictus and Aedes japonicus, Aedes koreicus has spread within Central Europe. Extensive information on its vector competence is missing. Ae. koreicus from Germany were investigated for their vector competence for chikungunya virus (CHIKV), Zika virus (ZIKV) and West Nile virus (WNV). Experiments were performed under different climate conditions (27 ± 5 °C; 24 ± 5 °C) for fourteen days. Ae. koreicus had the potential to transmit CHIKV and ZIKV but not WNV. Transmission was exclusively observed at the higher temperature, and transmission efficiency was rather low, at 4.6% (CHIKV) or 4.7% (ZIKV). Using a whole virome analysis, a novel mosquito-associated virus, designated Wiesbaden virus (WBDV), was identified in Ae. koreicus. Linking the WBDV infection status of single specimens to their transmission capability for the arboviruses revealed no influence on ZIKV transmission. In contrast, a coinfection of WBDV and CHIKV likely has a boost effect on CHIKV transmission. Due to its current distribution, the risk of arbovirus transmission by Ae. koreicus in Europe is rather low but might gain importance, especially in regions with higher temperatures. The impact of WBDV on arbovirus transmission should be analyzed in more detail.

Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps.

BACKGROUND: For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. METHODS: In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. RESULTS: A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. CONCLUSION: The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory.