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Abstract Background In recent years the Asian bush mosquito Aedes japonicus has invaded Europe, including the Netherlands. This species is a known vector for a range of arboviruses, possibly including West Nile virus (WNV). As WNV emerged in the Netherlands in 2020, it is important to investigate the vectorial capacity of mosquito species present in the Netherlands to estimate the risk of future outbreaks and further spread of the virus. Therefore, this study evaluates the potential role of Ae. japonicus in WNV transmission and spillover from birds to dead-end hosts in the Netherlands. Methods We conducted human landing collections in allotment gardens (Lelystad, the Netherlands) in June, August and September 2021 to study the diurnal and seasonal host-seeking behaviour of Ae. japonicus. Furthermore, their host preference in relation to birds using live chicken-baited traps was investigated. Vector competence of field-collected Ae. japonicus mosquitoes for two isolates of WNV at two different temperatures was determined. Based on the data generated from these studies, we developed a Susceptible-Exposed-Infectious-Recovered (SEIR) model to calculate the risk of WNV spillover from birds to humans via Ae. japonicus, under the condition that the virus is introduced and circulates in an enzootic cycle in a given area. Results Our results show that Ae. japonicus mosquitoes are actively host seeking throughout the day, with peaks in activity in the morning and evening. Their abundance in August was higher than in June and September. For the host-preference experiment, we documented a small number of mosquitoes feeding on birds: only six blood-fed females were caught over 4 full days of sampling. Finally, our vector competence experiments with Ae. japonicus compared to its natural vector Culex pipiens showed a higher infection and transmission rate when infected with a local, Dutch, WNV isolate compared to a Greek isolate of the virus. Interestingly, we also found a small number of infected Cx. pipiens males with virus-positive leg and saliva samples. Conclusions Combining the field and laboratory derived data, our model predicts that Ae. japonicus could act as a spillover vector for WNV and could be responsible for a high initial invasion risk of WNV when present in large numbers. Graphical Abstract

ABSTRACTTick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Abstract 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. Graphical Abstract

Abstract The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV.

Abstract Background Ixodes ricinus ticks are infected with a large diversity of vertically and horizontally transmitted symbionts. While horizontally transmitted symbionts rely on a vertebrate host for their transmission, vertically transmitted symbionts rely more on the survival of their invertebrate host for transmission. We therefore hypothesized horizontally transmitted symbionts to be associated with increased tick activity to increase host contact rate and vertically transmitted symbionts to be associated with higher tick weight and lipid fraction to promote tick survival. Methods We used a behavioural assay to record the questing activity of I. ricinus ticks. In addition, we measured weight and lipid fraction and determined the presence of ten symbiont species in these ticks using qPCR, of which six were vertically transmitted and four horizontally transmitted. Results Vertically transmitted symbionts (e.g. Midichloria mitochondrii) were associated with an increase in tick weight, whereas horizontally transmitted symbionts (e.g. Borrelia burgdorferi sensu lato) were often associated with lower weight and lipid fraction of ticks. Moreover, horizontally transmitted symbionts (e.g. B. burgdorferi s.l.) were associated with increased tick activity, which may benefit pathogen transmission and increases tick-borne disease hazard. Conclusions Our study shows that horizontally and vertically transmitted symbionts differentially influence the behaviour and physiology of I. ricinus and warrants future research to study the underlying mechanisms and effects on transmission dynamics of tick-borne pathogens. Graphical abstract

Rift Valley fever virus (RVFV) is a (re)emerging mosquito-borne pathogen impacting human and animal health. How RVFV spreads through a population depends on population-level and individual-level interactions between vector, host and pathogen. Here, we estimated the probability for RVFV to transmit to naive animals by experimentally exposing lambs to a bite of an infectious mosquito, and assessed if and how RVFV infection subsequently developed in the exposed animal. Aedes aegypti mosquitoes, previously infected via feeding on a viremic lamb, were used to expose naive lambs to the virus. Aedes aegypti colony mosquitoes were used as they are easy to maintain and readily feed in captivity. Other mosquito spp. could be examined with similar methodology. Lambs were exposed to either 1-3 (low exposure) or 7-9 (high exposure) infectious mosquitoes. All lambs in the high exposure group became viremic and showed characteristic signs of Rift Valley fever within 2-4 days post exposure. In contrast, 3 out of 12 lambs in the low exposure group developed viremia and disease, with similar peak-levels of viremia as the high exposure group but with some heterogeneity in the onset of viremia. These results suggest that the likelihood for successful infection of a ruminant host is affected by the number of infectious mosquitoes biting, but also highlights that a single bite of an infectious mosquito can result in disease. The per bite mosquito-to-host transmission efficiency was estimated at 28% (95% confidence interval: 15 - 47%). We subsequently combined this transmission efficiency with estimates for life traits of Aedes aegypti or related mosquitoes into a Ross-McDonald mathematical model to illustrate scenarios under which major RVFV outbreaks could occur in naïve populations (i.e., R0 >1). The model revealed that relatively high vector-to-host ratios as well as mosquitoes feeding preferably on competent hosts are required for R0 to exceed 1. Altogether, this study highlights the importance of experiments that mimic natural exposure to RVFV. The experiments facilitate a better understanding of the natural progression of disease and a direct way to obtain epidemiological parameters for mathematical models.

Abstract Background Tick-borne encephalitis virus (TBEV) can cause severe neurological disease in humans. Its geographical distribution is expanding in Western Europe with unresolved causes and spatial patterns, necessitating enhanced surveillance. Monitoring the virus in the environment is complicated, as it usually relies on destructive sampling of small rodents to test organs for TBEV, which in addition to ethical considerations also raises issues for long-term monitoring or longitudinal studies. Moreover, even when the virus is not detected in the blood or organs of the rodent, TBEV can still be transmitted from an infected tick to uninfected ticks feeding nearby. This is due to the ability of TBEV to replicate and migrate locally within the epidermis of small mammals, including those that do not appear to have systemic infection. This suggests that the virus may be detectable in skin biopsies, which has been confirmed in experimentally infected laboratory rodents, but it remains unknown if this sample type may be a viable alternative to destructively obtained samples in the monitoring of natural TBEV infection. Here we test ear tissue and dried blood spot (DBS) samples from rodents to determine whether TBEV-RNA can be detected in biological samples obtained non-destructively. Methods Rodents were live-trapped and sampled at three woodland areas in The Netherlands where presence of TBEV has previously been recorded. Ear tissue (n = 79) and DBSs (n = 112) were collected from a total of 117 individuals and were tested for TBEV-RNA by real-time RT-PCR. Results TBEV-RNA was detected in five rodents (4.3% of tested individuals), all of which had a TBEV-positive ear sample, while only two out of four of these individuals (for which a DBS was available) had a positive DBS. This equated to 6.3% of ear samples and 1.8% of DBSs testing positive for TBEV-RNA. Conclusions We provide the first evidence to our knowledge that TBEV-RNA can be detected in samples obtained non-destructively from naturally infected wild rodents, providing a viable sampling alternative suitable for longitudinal surveillance of the virus. Graphical Abstract

Abstract Background Evidence suggests a vicious cycle between rice cultivation and malaria control in Rwanda. Rice fields offer an attractive breeding ground for malaria vectors, which increases the disease burden in rice farming communities, and, consequently, reduces productivity in the rice sector. Community-based larval source management in rice fields is propagated as a sustainable solution to break this cycle. A sense of agency and ownership of malaria control interventions, as well as the mobilization of resources at the local level, are often considered preconditions for success. However, an evidence gap exists regarding the interaction between the agentive and financial dimension of local sustainability. Methods We conduct a larviciding pilot involving three groups; one group where rice farmers sprayed their fields under expert supervision, one group where rice farmers organised the larviciding campaign themselves, and a (non-sprayed) control group. We test whether the difference in agency between the intervention groups affects farmers’ willingness-to-pay for a larviciding campaign. Willingness-to-pay is elicited in a contingent valuation exercise, more specifically a bidding game, and is assessed both before and after the pilot (n = 288). Difference-in-difference estimates are computed, using a propensity score matching technique. Supplementary data were collected in a survey and two focus group discussions for triangulation. Results The high-agency (self-organised) group significantly outperforms the low-agency (expert-supervised) group in terms of maintaining its willingness to contribute financially. However, higher willingness-to-pay in the high-agency group does not appear to be driven by a stronger sense of ownership per se. The supplementary data indicate high levels of ownership in both treatment groups compared to the control group. A tentative explanation lies in diverging perceptions concerning the effectiveness of the pilot. Conclusions The study supports the idea that community-led organization of larval source management can prove instrumental in mobilizing finance for malaria control in low-income settings where rice production interferes with the fight against malaria. However, the causality is complex. Feelings of ownership do not appear the main driver of willingness-to-pay, at least not directly, which opens up the possibility of initiating community-driven malaria control interventions that promote the agentive and financial dimension of local sustainability simultaneously.

Abstract Mosquito species belonging to the genus Aedes have attracted the interest of scientists and public health officers because of their capacity to transmit viruses that affect humans. Some of these species were brought outside their native range by means of trade and tourism and then colonised new regions thanks to a unique combination of eco-physiological traits. Considering mosquito physiological and behavioural traits to understand and predict their population dynamics is thus a crucial step in developing strategies to mitigate the local densities of invasive Aedes populations. Here, we synthesised the life cycle of four invasive Aedes species (Ae. aegypti, Ae. albopictus, Ae. japonicus and Ae. koreicus) in a single multi-scale stochastic modelling framework which we coded in the R package dynamAedes. We designed a stage-based and time-discrete stochastic model driven by temperature, photo-period and inter-specific larval competition that can be applied to three different spatial scales: punctual, local and regional. These spatial scales consider different degrees of spatial complexity and data availability by accounting for both active and passive dispersal of mosquito species as well as for the heterogeneity of the input temperature data. Our overarching aim was to provide a flexible, open-source and user-friendly tool rooted in the most updated knowledge on the species’ biology which could be applied to the management of invasive Aedes populations as well as to more theoretical ecological inquiries. Graphical Abstract

Zika virus (ZIKV), the causative agent of virus-induced brain damage in newborns, is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus. Here, Obadia et al. characterize ZIKV vector competence of 50 mosquito populations from six species collected in 12 different countries to inform about epidemic risk. They find that African ZIKV strain shows higher transmission efficiency compared to American and Asian ZIKV strains and that Ae. aegypti mosquitoes have highest susceptibility to infections, while Culexmosquitoes are largely non-susceptible.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.

Abstract Background As part of malaria prevention and control efforts, the distribution and density of malaria mosquitoes requires continuous monitoring. Resources for long-term surveillance of malaria vectors, however, are often limited. The aim of the research was to evaluate the value of citizen science in providing insight into potential malaria vector hotspots and other malaria relevant information, and to determine predictors of malaria vector abundance in a region where routine mosquito monitoring has not been established to support vector surveillance. Methods A 1-year citizen science programme for malaria mosquito surveillance was implemented in five villages of the Ruhuha sector in Bugesera district, Rwanda. In total, 112 volunteer citizens were enrolled and reported monthly data on mosquitoes collected in their peridomestic environment using handmade carbon-dioxide baited traps. Additionally, they reported mosquito nuisance experienced as well as the number of confirmed malaria cases in their household. Results In total, 3793 female mosquitoes were collected, of which 10.8% were anophelines. For the entire period, 16% of the volunteers reported having at least one confirmed malaria case per month, but this varied by village and month. During the study year 66% of the households reported at least one malaria case. From a sector perspective, a higher mosquito and malaria vector abundance was observed in the two villages in the south of the study area. The findings revealed significant positive correlations among nuisance reported and confirmed malaria cases, and also between total number of Culicidae and confirmed malaria cases, but not between the numbers of the malaria vector Anopheles gambiae and malaria cases. At the sector level, of thirteen geographical risk factors considered for inclusion in multiple regression, distance to the river network and elevation played a role in explaining mosquito and malaria mosquito abundance. Conclusions The study demonstrates that a citizen science approach can contribute to mosquito monitoring, and can help to identify areas that, in view of limited resources for control, are at higher risk of malaria.

Abstract Background Infectious blood meal experiments have been frequently performed with different virus-vector combinations to assess the transmission potential of arthropod-borne (arbo)viruses. A wide variety of host blood sources have been used to deliver arboviruses to their arthropod vectors in laboratory studies. The type of blood used during vector competence experiments does not always reflect the blood from the viremic vertebrate hosts in the field, but little is known about the effect of blood source on the experimental outcome of vector competence studies. Here we investigated the effect of avian versus human blood on the infection and transmission rates of the zoonotic Usutu virus (USUV) in its primary mosquito vector Culex pipiens. Methods Cx. pipiens biotypes (pipiens and molestus) were orally infected with USUV through infectious blood meals containing either chicken or human whole blood. The USUV infection and transmission rates were determined by checking mosquito bodies and saliva for USUV presence after 14 days of incubation at 28 °C. In addition, viral titers were determined for USUV-positive mosquito bodies and saliva. Results Human and chicken blood lead to similar USUV transmission rates for Cx. pipiens biotype pipiens (18% and 15%, respectively), while human blood moderately but not significantly increased the transmission rate (30%) compared to chicken blood (17%) for biotype molestus. USUV infection rates with human blood were consistently higher in both Cx. pipiens biotypes compared to chicken blood. In virus-positive mosquitoes, USUV body and saliva titers did not differ between mosquitoes taking either human or chicken blood. Importantly, biotype molestus had much lower USUV saliva titers compared to biotype pipiens, regardless of which blood was offered. Conclusions Infection of mosquitoes with human blood led to higher USUV infection rates as compared to chicken blood. However, the blood source had no effect on the vector competence for USUV. Interestingly, biotype molestus is less likely to transmit USUV compared to biotype pipiens due to very low virus titers in the saliva.

Abstract Background Many countries, including Rwanda, have mosquito monitoring programmes in place to support decision making in the fight against malaria. However, these programmes can be costly, and require technical (entomological) expertise. Involving citizens in data collection can greatly support such activities, but this has not yet been thoroughly investigated in a rural African context. Methods Prior to the implementation of such a citizen-science approach, a household entomological survey was conducted in October–November 2017 and repeated one year later in Busoro and Ruhuha sectors, in southern and eastern province of Rwanda, respectively. The goal was to evaluate the perception of mosquito nuisance reported by citizens as a potential indicator for malaria vector hotspots. Firstly, mosquito abundance and species composition were determined using Centers for Disease Control and Prevention (CDC) light traps inside the houses. Secondly, household members were interviewed about malaria risk factors and their perceived level of mosquito nuisance. Results Tiled roofs, walls made of mud and wood, as well as the number of occupants in the house were predictors for the number of mosquitoes (Culicidae) in the houses, while the presence of eaves plus walls made of mud and wood were predictors for malaria vector abundance. Perception of mosquito nuisance reported indoors tended to be significantly correlated with the number of Anopheles gambiae sensu lato (s.l.) and Culicidae collected indoors, but this varied across years and sectors. At the village level, nuisance also significantly correlated with An. gambiae s.l. and total mosquito density, but only in 2018 while not in 2017. Conclusions Perception of mosquito nuisance denoted in a questionnaire survey could be used as a global indicator of malaria vector hotspots. Hence, involving citizens in such activities can complement malaria vector surveillance and control.

Abstract Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.

Abstract Bluetongue virus (BTV) serotype 8 has been circulating in Europe since a major outbreak occurred in 2006, causing economic losses to livestock farms. The unpredictability of the biting activity of midges that transmit BTV implies difficulty in computing accurate transmission models. This study uniquely integrates field collections of midges at a range of European latitudes (in Sweden, The Netherlands, and Italy), with a multi-scale modelling approach. We inferred the environmental factors that influence the dynamics of midge catching, and then directly linked predicted midge catches to BTV transmission dynamics. Catch predictions were linked to the observed prevalence amongst sentinel cattle during the 2007 BTV outbreak in The Netherlands using a dynamic transmission model. We were able to directly infer a scaling parameter between daily midge catch predictions and the true biting rate per cow per day. Compared to biting rate per cow per day the scaling parameter was around 50% of 24 h midge catches with traps. Extending the estimated biting rate across Europe, for different seasons and years, indicated that whilst intensity of transmission is expected to vary widely from herd to herd, around 95% of naïve herds in western Europe have been at risk of sustained transmission over the last 15 years.

Abstract Background Malaria control remains a challenge globally and in malaria-endemic countries in particular. In Rwanda, a citizen science programme has been set up to improve malaria control. Citizens are involved in collecting mosquito species and reporting mosquito nuisance. This study assessed what people benefit from such a citizen science programme. The analysis was conducted on how the citizen science programme influenced perceptions and behaviour related to malaria control. Methods This study employed a mixed-methods approach using dissemination workshops, a survey, and village meetings as the main data collection methods. Dissemination workshops and village meetings involved 112 volunteers of the citizen science programme and were conducted to explore: (1) the benefits of being involved in the programme and (2) different ways used to share malaria-related information to non-volunteers. The survey involved 328 people (110 volunteers and 218 non-volunteers) and was used to compare differences in malaria-related perceptions and behaviour over time (between 2017 and 2019), as well as between volunteers and non-volunteers. Results Malaria-related perceptions and behaviour changed significantly over time (between 2017 and 2019) and became favourable to malaria control. When the findings were compared between volunteers and non-volunteers, for perceptions, only perceived self-efficacy showed a significant difference between these two groups. However, volunteers showed significantly more social interaction, participation in malaria-related activities at the community level, and indoor residual spraying (IRS) acceptance. In addition, both volunteers and non-volunteers reported to have gained knowledge and skills about the use of malaria control measures in general, and mosquito species in particular among volunteers. Conclusion The reported knowledge and skills gained among non-volunteers indicate a diffusion of the citizen science programme-related information in the community. Thus, the citizen science programme has the potential to provide individual and collective benefits to volunteers and society at large.

Abstract Background To further reduce malaria, larval source management (LSM) is proposed as a complementary strategy to the existing strategies. LSM has potential to control insecticide resistant, outdoor biting and outdoor resting vectors. Concerns about costs and operational feasibility of implementation of LSM at large scale are among the reasons the strategy is not utilized in many African countries. Involving communities in LSM could increase intervention coverage, reduce costs of implementation and improve sustainability of operations. Community acceptance and participation in community-led LSM depends on a number of factors. These factors were explored under the Majete Malaria Project in Chikwawa district, southern Malawi. Methods Separate focus group discussions (FGDs) were conducted with members from the general community (n = 3); health animators (HAs) (n = 3); and LSM committee members (n = 3). In-depth interviews (IDIs) were conducted with community members. Framework analysis was employed to determine the factors contributing to community acceptance and participation in the locally-driven intervention. Results Nine FGDs and 24 IDIs were held, involving 87 members of the community. Widespread knowledge of malaria as a health problem, its mode of transmission, mosquito larval habitats and mosquito control was recorded. High awareness of an association between creation of larval habitats and malaria transmission was reported. Perception of LSM as a tool for malaria control was high. The use of a microbial larvicide as a form of LSM was perceived as both safe and effective. However, actual participation in LSM by the different interviewee groups varied. Labour-intensiveness and time requirements of the LSM activities, lack of financial incentives, and concern about health risks when wading in water bodies contributed to lower participation. Conclusion Community involvement in LSM increased local awareness of malaria as a health problem, its risk factors and control strategies. However, community participation varied among the respondent groups, with labour and time demands of the activities, and lack of incentives, contributing to reduced participation. Innovative tools that can reduce the labour and time demands could improve community participation in the activities. Further studies are required to investigate the forms and modes of delivery of incentives in operational community-driven LSM interventions.

Abstract Background Application of the larvicide Bacillus thuringiensis var. israelensis (Bti) is a viable complementary strategy for malaria control. Efficacy of Bti is dose-dependent. There is a knowledge gap on the effects of larval exposure to sublethal Bti doses on emerging adult mosquitoes. The present study examined the effect of larval exposure to sublethal doses of Bti on the survival, body size and oviposition rate in adult Anopheles coluzzii. Methods Third-instar An. coluzzii larvae were exposed to control and sublethal Bti concentrations at LC20, LC50 and LC70 for 48 h. Surviving larvae were reared to adults under standard colony conditions. Thirty randomly selected females from each treatment were placed in separate cages and allowed to blood feed. Twenty-five gravid females from the blood-feeding cages were randomly selected and transferred into new cages where they were provided with oviposition cups. Numbers of eggs laid in each cage and mortality of all adult mosquitoes were recorded daily. Wing lengths were measured of 570 mosquitoes as a proxy for body size. Results Exposure to LC70 Bti doses for 48 h as third-instar larvae reduced longevity of adult An. coluzzii mosquitoes. Time to death was 2.58 times shorter in females exposed to LC70 Bti when compared to the control females. Estimated mortality hazard rates were also higher in females exposed to the LC50 and LC20 treatments, but these differences were not statistically significant. The females exposed to LC70 concentrations had 12% longer wings than the control group (P < 0.01). No differences in oviposition rate of the gravid females were observed between the treatments. Conclusions Exposure of An. coluzzii larvae to sublethal Bti doses reduces longevity of resultant adults and is associated with larger adult size and unclear effect on oviposition. These findings suggest that anopheline larval exposure to sublethal Bti doses, though not recommended, could reduce vectorial capacity for malaria vector populations by increasing mortality of resultant adults.

Abstract Background In the Netherlands, Aedes albopictus has been found each year since 2010 during routine exotic mosquito species surveillance at companies that import used tires. We developed habitat suitability models to investigate the potential risk of establishment and spread of this invasive species at these locations. Methods We used two methodologies: first, a species distribution model based on the maximum entropy modelling approach (MaxEnt) taking into consideration updated occurrence data of the species in Europe, and secondly, a spatial logic conditional model based on the temperature requirements of the species and using land surface temperature data (LST model). Results Suitability assessment obtained with the MaxEnt model at European level accurately reflect the current distribution of the species and these results also depict moderately low values in parts of the Netherlands, Belgium, Denmark, the British islands and southern parts of Scandinavia. Winter temperature was the variable that contributed most to the performance of the model (47.3%). The results of the LST model showed that: (i) coastal areas are suitable for overwintering of eggs; (ii) large areas in the northern part of the country have a low suitability for adult survival; and (iii) the entire country is suitable for successful completion of the life-cycle if the species is introduced after the winter months. Results of the LST model revealed that temperatures in 2012 and 2014 did not limit the overwintering of eggs or survival of adults at the locations where the species was found. By contrast, for the years 2010, 2011 and 2013, overwintering of eggs at these locations is considered unlikely. Conclusions Results using two modelling methodologies show differences in predicted habitat suitability values. Based on the results of both models, the climatic conditions could hamper the successful overwintering of eggs of Ae. albopictus and their survival as adults in many areas of the country. However, during warm years with mild winters, many areas of the Netherlands offer climatic conditions suitable for developing populations. Regular updates of the models, using updated occurrence and climatic data, are recommended to study the areas at risk.

Abstract Background Emerging arboviral diseases like Zika, dengue and chikungunya that are transmitted by Aedes aegypti mosquitoes, are increasingly threatening human health. Blends of human-like synthetic chemical attractants can be used to attract host-seeking mosquitoes. The aim of this study was to test new combinations of traps and odour baits in the laboratory, followed by testing the best candidates in the field to improve Ae. aegypti monitoring and surveillance. Methods First, the BG-Suna trap was evaluated for capturing laboratory-reared Ae. aegypti by testing normal and inverted positions in screen cage tests. Secondly, the attractiveness of the MB5 blend, CO2, and their combination was tested. Thirdly, we tested the attractiveness of different trap types (BG-Suna, BG-Sentinel, MM-X and CDC light trap). Finally, we confirmed laboratory results in the field in Paramaribo, Suriname, using the MB5 and BG-Lure odour blends, CO2 and the BG-Sentinel and BG-Bowl trap using a Latin Square design. Results The MB5 blend in combination with CO2 outperformed traps baited only with CO2 or MB5 in screen cage tests (P

ABSTRACTUsutu virus (USUV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that are mainly transmitted between bird hosts by vector mosquitoes. Infections in humans are incidental but can cause severe disease. USUV is endemic in large parts of Europe, while WNV mainly circulates in Southern Europe. In recent years, WNV is also frequently detected in Northern Europe, thereby expanding the area where both viruses co-circulate. However, it remains unclear how USUV may affect the future spread of WNV and the likelihood of human co-infection. Here we investigated whether co-infections with both viruses in cell lines and their primary mosquito vector, Culex pipiens, affect virus replication and transmission dynamics. We show that USUV is outcompeted by WNV in mammalian, avian and mosquito cells during co-infection. Mosquitoes that were exposed to both viruses simultaneously via infectious blood meal displayed significantly reduced USUV transmission compared to mosquitoes that were only exposed to USUV (from 15% to 3%), while the infection and transmission of WNV was unaffected. In contrast, when mosquitoes were pre-infected with USUV via infectious blood meal, WNV transmission was significantly reduced (from 44% to 17%). Injection experiments established the involvement of the midgut in the observed USUV-mediated WNV inhibition. The competition between USUV and WNV during co-infection clearly indicates that the chance of concurrent USUV and WNV transmission via a single mosquito bite is low. The competitive relation between USUV and WNV may impact virus transmission dynamics in the field and affect the epidemiology of WNV in Europe.

Ticks and tick-borne diseases cause substantial economic losses to the livestock industry in sub-Saharan Africa. Mazao Tickoff is a novel bioacaricide developed for tick control and is based on the entomopathogenic fungus Metarhizium anisopliae sensu lato (s.l.) isolate ICIPE 7. To date, no randomized controlled study has been undertaken to demonstrate the efficacy of this bioacaricide in reducing natural tick infestation on cattle. To this end, this field trial is designed to evaluate the anti-tick efficacy of Mazao Tickoff on cattle in coastal Kenya compared to a standard chemical tick control protocol. In this prospective, multi-center randomized controlled trial, eligible herds will be randomized by the herd size to the intervention arm in a 1:1:1 ratio to either Triatix® (active ingredient: amitraz); Mazao Tickoff (active ingredient: M. anisopliae ICIPE 7); or placebo (excipients of the Mazao Tickoff), with a total enrollment target of 1,077 cattle. Treatments will be dispensed on Day 0 (defined individually as the day each animal receives the first treatment) and thereafter every two weeks until Day 182. Ticks will be counted on every animal in each herd (herds to be included have at least one animal bearing at least one tick on Day 0), and thereafter on bi-weekly intervals until Day 182. The primary efficacy assessments of Mazao Tickoff will be based on the mean percentage reduction in tick counts at each post-treatment follow-up visit compared to the placebo group and the Triatix® arm. Further, the effect of Mazao Tickoff on the prevalence of common cattle pathogens, Anaplasma marginale and Theileria parva, will be determined by assessing incidence and seroprevalence at four different time points. This protocol describes the first rigorous evaluation of the efficacy of Mazao Tickoff and its potential as a viable alternative non-chemical acaricide tool for tick control in Kenya and elsewhere.

For effective sampling of mosquitoes in malaria surveillance programmes, it is essential to include attractive cues in traps. With the aim of implementing a citizen science project on malaria vectors in rural Rwanda, a handmade plastic bottle trap was designed and tested in the field to determine its effectiveness in capturing adult Anopheles gambiae sensu lato, the main malaria vector, and other mosquito species. Carbon dioxide (CO2) and light were used as attractive cues. CO2 was produced by inoculating sugar with yeast and water. Light was emitted from a torch by light-emitting diodes (LEDs). Under field conditions in rural Rwanda, three handmade trap designs were compared to Centers for Disease Control and Prevention miniature light traps (CDC-LT) in houses. The trap baited with yeast produced CO2 and light caught the highest number of mosquitoes compared to the traps baited with light alone or CO2 alone. The number of An. gambiae s.l. in the handmade trap with light and CO2 was approximately 9–10% of the number caught with a CDC light trap. This suggests that about 10 volunteers with a handmade trap could capture a similar-sized sample of An. gambiae as one CDC-LT would collect. Based on these findings, the handmade plastic bottle trap baited with sugar fermenting yeast and light represents an option for inclusion in mosquito surveillance activities in a citizen science context.

Abstract Background Malaria preventive measures, including long-lasting insecticide-treated bet nets (LLINs), indoor residual spraying (IRS), and controlling mosquito breeding sites, are key measures to achieve malaria elimination. Still, compliance with these recommended measures remains a major challenge. By applying a novel and comprehensive model for determinants of malaria prevention behaviour, this study tests how individual perceptions influence the intentions to use malaria preventive measures and explores strategies that stimulate their consistent use. Methods The study was carried out in the sectors of Ruhuha and Busoro, Rwanda during October and November 2017, and these were conducted into two phases. Phase one involved a questionnaire survey (N = 742), whereas Phase two employed a qualitative approach that included nine focus group discussions, seven key informant interviews, and three in-depth interviews. Results The findings of the quantitative study showed that participants very often use LLINs (66.6%), accept IRS (73.9%), and drain stagnant water in case of presence (62%). The intentions to use malaria preventive measures were consistently driven by perceived severity, perceived self-efficacy, perceived response efficacy, and subjective norms, and hindered by perceived barriers. The intentions were also positively associated with the actual use of LLINs, acceptance of IRS, and drainage of stagnant water. There is no evidence that either not having enough LLINs (ownership of at least one bed net in the household, here referred to as availability) or having sufficient LLINs (having one LLIN per two people in the household, here referred to as accessibility) moderated the relationship between behavioural intentions and actual use of LLINs. The qualitative study indicated that participants believed malaria risk to be high and perceived a high mosquito density. They also believed that repetitive malaria episodes are caused by the perceived low effectiveness of anti-malaria medications. Lack of LLINs increased the perceived added value of LLINs, and together with the increased malaria burden increased the perceived response efficacy. Participants highlighted the need to continuously mobilize and engage community members especially those who do not use LLINs when having one, and those who do not accept the spraying activities. Conclusion Malaria prevention interventions should target individual perceptions to enhance consistent use of malaria preventive measures. Three strategies to improve consistent use and acceptance of these measures are highlighted: (1) ensure access to LLINs and regular spraying activities, (2) community mobilization and (3) citizen engagement in malaria prevention activities.

Abstract Background West Nile virus (WNV) is a mosquito-borne virus that is mainly transmitted among birds by Culex pipiens mosquitoes. The species Cx. pipiens consists of two biotypes named pipiens and molestus, which together can form hybrids. One of the major distinctions between the biotypes is their overwintering behaviour. Adults of biotype pipiens diapause during winter, whereas biotype molestus remains actively blood-feeding. Diapausing may affect survival and vector competence of biotype pipiens. The aims of this study were therefore to identify the biotype composition of diapausing Cx. pipiens mosquitoes, to quantify survival throughout the autumn and winter months, and to determine effects of overwintering on vector competence of emerging Cx. pipiens mosquitoes for WNV. Methods Diapausing mosquitoes were collected at two typical overwintering locations in the Netherlands. A selection of Cx. pipiens mosquitoes was identified to biotype using real-time PCR. Survival of diapausing Cx. pipiens mosquitoes during autumn and winter was monitored by placing cages with either field-collected or laboratory-reared females in houses and sheds. Vector competence of field-collected (diapausing) and laboratory-reared (non-diapausing) Cx. pipiens mosquitoes was determined to gain insight in the effect of overwintering on WNV transmission. Results The majority (92%) of diapausing Cx. pipiens females were identified as biotype pipiens. More than 70% of diapausing Cx. pipiens mosquitoes was able to survive for more than four months in sheds, whereas diapausing in houses resulted in 100% mortality in that same period. In contrast, non-diapausing Cx. pipiens biotype pipiens mosquitoes reared in the laboratory died within a week in both houses and sheds. Vector competence of Cx. pipiens mosquitoes that had diapaused during the autumn and winter months was comparable to non-diapausing laboratory-reared mosquitoes. Conclusions This study confirms that the majority of Cx. pipiens mosquitoes in their typical overwintering site belongs to the pipiens biotype. It shows that more than two-third of diapausing Cx. pipiens mosquitoes is able to survive winter under sheltered winter conditions. Finally, vector competence for WNV of mosquitoes that emerge from overwintering sites is not affected by their relatively old age.

Despite their past importance as vectors of indigenous malaria, the species composition and spatial distribution of the members of the Anopheles maculipennis complex have been studied to a limited extent in the Netherlands. Therefore, this investigation focuses on the distribution of the members of this complex in the Netherlands, including Anopheles daciae, which has recently been found in countries bordering the Netherlands. In the framework of a national mosquito surveillance between 2010 and 2021, a total of 541 specimens of An. maculipennis s.l. were analyzed from 161 locations covering the entire territory. In addition, 89 specimens were analyzed from overwintering sites during the winter of 2020/2021. All individual mosquitoes were identified to species-level using Sanger sequencing of the ribosomal internal transcribed spacer 2. To characterize the habitat of An. maculipennis s.l. in the Netherlands, land cover use data was extracted in a 1 km buffer area around each finding location. For populations collected in summers between 2010 and 2021, the most frequent species was An. messeae, present in 88.19% of the locations, followed by An. maculipennis s.s. (11.80%), An. atroparvus (3.72%) and An. daciae (3.72%). Anopheles daciae was found in the southern inland areas of the country. Furthermore, An. messeae and An. daciae occurred in sympatry at overwintering sites. This study provides relevant information on the occurrence of species of the Anopheles maculipennis complex in the Netherlands, contributing to a better estimation of the risk of mosquito-borne disease in the country.

Abstract Background The outbreaks of bluetongue and Schmallenberg disease in Europe have increased efforts to understand the ecology of Culicoides biting midges and their role in pathogen transmission. However, most studies have focused on a specific habitat, region, or country. To facilitate wider comparisons, and to obtain a better understanding of the spread of disease through Europe, the present study focused on monitoring biting midge species diversity in three different habitat types and three countries across Europe. Methods Biting midges were trapped using Onderstepoort Veterinary Institute light traps at a total of 27 locations in Sweden, the Netherlands and Italy, comprising farm, peri-urban and wetland habitats. From July 2014 to June 2015 all locations were sampled monthly, except for during the winter months. Trapped midges were counted and identified morphologically. Indices on species richness, evenness and diversity were calculated. Community compositions were analysed using non-metric multidimensional scaling (NMDS) techniques. Results A total of 50,085 female midges were trapped during 442 collection nights. More than 88% of these belonged to the Obsoletus group. The highest midge diversity was found in Sweden, while species richness was highest in the Netherlands, and most specimens were trapped in Italy. For habitats within countries, diversity of the trapped midges was lowest for farms in all countries. Differences in biting midge species communities were more distinct across the three countries than the three habitat types. Conclusions A core midge community could be identified, in which the Obsoletus group was the most abundant. Variations in vector communities across countries imply different patterns of disease spread throughout Europe. How specific species and their associated communities affect disease risk is still unclear. Our results emphasize the importance of midge diversity data at community level, how this differs across large geographic range within Europe, and its implications on assessing risks of midge-borne disease outbreaks.

Abstract Background Studies on mosquito species diversity in Europe often focus on a specific habitat, region or country. Moreover, different trap types are used for these sampling studies, making it difficult to compare and validate results across Europe. To facilitate comparisons of trapping sites and community analysis, the present study used two trap types for monitoring mosquito species diversity in three habitat types for three different countries in Europe. Methods Mosquitoes were trapped using Biogents Sentinel (BGS), and Mosquito Magnet Liberty Plus (MMLP) traps at a total of 27 locations in Sweden, the Netherlands and Italy, comprising farm, peri-urban and wetland habitats. From July 2014 to June 2015 all locations were sampled monthly, except for the winter months. Indices of species richness, evenness and diversity were calculated, and community analyses were carried out with non-metric multidimensional scaling (NMDS) techniques. Results A total of 11,745 female mosquitoes were trapped during 887 collections. More than 90% of the mosquitoes belonged to the genera Culex and Aedes, with Culex pipiens being the most abundant species. The highest mosquito diversity was found in Sweden. Within Sweden, species diversity was highest in wetland habitats, whereas in the Netherlands and Italy this was highest at farms. The NMDS analyses showed clear differences in mosquito communities among countries, but not among habitat types. The MMLP trapped a higher diversity of mosquito species than the BGS traps. Also, MMLP traps trapped higher numbers of mosquitoes, except for the genera Culex and Culiseta in Italy. Conclusions A core mosquito community could be identified for the three countries, with Culex pipiens as the most abundant species. Differences in mosquito species communities were more defined by the three countries included in the study than by the three habitat types. Differences in mosquito community composition across countries may have implications for disease emergence and further spread throughout Europe. Future research should, therefore, focus on how field data of vector communities can be incorporated into models, to better assess the risk of mosquito-borne disease outbreaks.

Abstract Background Targeting the aquatic stages of malaria vectors via larval source management (LSM) in collaboration with local communities could accelerate progress towards malaria elimination when deployed in addition to existing vector control strategies. However, the precise role that communities can assume in implementing such an intervention has not been fully investigated. This study investigated community awareness, acceptance and participation in a study that incorporated the socio-economic and entomological impact of LSM using Bacillus thuringiensis var. israelensis (Bti) in eastern Rwanda, and identified challenges and recommendations for future scale-up. Methods The implementation of the community-based LSM intervention took place in Ruhuha, Rwanda, from February to July 2015. The intervention included three arms: control, community-based (CB) and project-supervised (PS). Mixed methods were used to collect baseline and endline socio-economic data in January and October 2015. Results A high perceived safety and effectiveness of Bti was reported at the start of the intervention. Being aware of malaria symptoms and perceiving Bti as safe on other living organisms increased the likelihood of community participation through investment of labour time for Bti application. On the other hand, the likelihood for community participation was lower if respondents: (1) perceived rice farming as very profitable; (2) provided more money to the cooperative as a capital; and, (3) were already involved in rice farming for more than 6 years. After 6 months of implementation, an increase in knowledge and skills regarding Bti application was reported. The community perceived a reduction in mosquito density and nuisance biting on treated arms. Main operational, seasonal and geographical challenges included manual application of Bti, long working hours, and need for transportation for reaching the fields. Recommendations were made for future scale-up, including addressing above-mentioned concerns and government adoption of LSM as part of its vector control strategies. Conclusions Community awareness and support for LSM increased following Bti application. A high effectiveness of Bti in terms of reduction of mosquito abundance and nuisance biting was perceived. The study confirmed the feasibility of community-based LSM interventions and served as evidence for future scale-up of Bti application and adoption into Rwandan malaria vector control strategies.

Abstract West Nile virus (WNV) is a mosquito-borne flavivirus which has caused repeated outbreaks in humans in southern and central Europe, but thus far not in northern Europe. The main mosquito vector for WNV, Culex pipiens, consists of two behaviourally distinct biotypes, pipiens and molestus, which can form hybrids. Differences between biotypes, such as vector competence and host preference, could be important in determining the risk of WNV outbreaks. Risks for WNV establishment can be modelled with basic reproduction number (R 0) models. However, existing R 0 models have not differentiated between biotypes. The aim of this study was, therefore, to explore the role of temperature-dependent and biotype-specific effects on the risk of WNV establishment in Europe. We developed an R 0 model with temperature-dependent and biotype-specific parameters, and calculated R 0 values using the next-generation matrix for several scenarios relevant for Europe. In addition, elasticity analysis was done to investigate the contribution of each biotype to R 0. Global warming and increased mosquito-to-host ratios can possibly result in more intense WNV circulation in birds and spill-over to humans in northern Europe. Different contributions of the Cx. pipiens biotypes to R 0 shows the importance of including biotype-specific parameters in models for reliable WNV risk assessments.

Zika virus (ZIKV) is a mosquito-borne pathogen that caused a large outbreak in the Americas in 2015 and 2016. The virus is currently present in tropical areas around the globe and can cause severe disease in humans, including Guillain-Barré syndrome and congenital microcephaly. The tropical yellow fever mosquito, Aedes aegypti, is the main vector in the urban transmission cycles of ZIKV. The discovery of ZIKV in wild-caught Culex mosquitoes and the ability of Culex quinquefasciatus mosquitoes to transmit ZIKV in the laboratory raised the question of whether the common house mosquito Culex pipiens, which is abundantly present in temperate regions in North America, Asia and Europe, could also be involved in ZIKV transmission. In this study, we investigated the vector competence of Cx. pipiens (biotypes molestus and pipiens) from the Netherlands for ZIKV, using Usutu virus as a control. After an infectious blood meal containing ZIKV, none of the tested mosquitoes accumulated ZIKV in the saliva, although 2% of the Cx. pipiens pipiens mosquitoes showed ZIKV–positive bodies. To test the barrier function of the mosquito midgut on virus transmission, ZIKV was forced into Cx. pipiens mosquitoes by intrathoracic injection, resulting in 74% (molestus) and 78% (pipiens) ZIKV–positive bodies. Strikingly, 14% (molestus) and 7% (pipiens) of the tested mosquitoes accumulated ZIKV in the saliva after injection. This is the first demonstration of ZIKV accumulation in the saliva of Cx. pipiens upon forced infection. Nevertheless, a strong midgut barrier restricted virus dissemination in the mosquito after oral exposure and we, therefore, consider Cx. pipiens as a highly inefficient vector for ZIKV.

For the development of sustainable control of tick-borne diseases, insight is needed in biological factors that affect tick populations. Here, the ecological interactions among Ixodiphagus hookeri, Ixodes ricinus, and two vertebrate species groups were investigated in relation to their effects on tick-borne disease risk. In 1129 questing ticks, I. hookeri DNA was detected more often in I. ricinus nymphs (4.4%) than in larvae (0.5%) and not in adults. Therefore, we determined the infestation rate of I. hookeri in nymphs from 19 forest sites, where vertebrate, tick, and tick-borne pathogen communities had been previously quantified. We found higher than expected co-occurrence rates of I. hookeri with deer-associated Anaplasma phagocytophilum, and lower than expected rates with rodent-associated Borrelia afzelii and Neoehrlichia mikurensis. The prevalence of I. hookeri in nymphs varied between 0% and 16% and was positively correlated with the encounter probability of ungulates and the densities of all life stages of I. ricinus. Lastly, we investigated the emergence of I. hookeri from artificially fed, field-collected nymphs. Adult wasps emerged from seven of the 172 fed nymphs. From these observations, we inferred that I. hookeri is parasitizing I. ricinus larvae that are feeding on deer, rather than on rodents or in the vegetation. Since I. hookeri populations depend on deer abundance, the main propagation host of I. ricinus, these wasps have no apparent effect on tick populations. The presence of I. hookeri may directly interfere with the transmission cycle of A. phagocytophilum, but not with that of B. afzelii or N. mikurensis.

Mosquitoes are vectors of arboviruses affecting animal and human health. Arboviruses circulate primarily within an enzootic cycle and recurrent spillovers contribute to the emergence of human-adapted viruses able to initiate an urban cycle involving anthropophilic mosquitoes. The increasing volume of travel and trade offers multiple opportunities for arbovirus introduction in new regions. This scenario has been exemplified recently with the Zika pandemic. To incriminate a mosquito as vector of a pathogen, several criteria are required such as the detection of natural infections in mosquitoes. In this study, we used a high-throughput chip based on the BioMark™ Dynamic arrays system capable of detecting 64 arboviruses in a single experiment. A total of 17,958 mosquitoes collected in Zika-endemic/epidemic countries (Brazil, French Guiana, Guadeloupe, Suriname, Senegal, and Cambodia) were analyzed. Here we show that this new tool can detect endemic and epidemic viruses in different mosquito species in an epidemic context. Thus, this fast and low-cost method can be suggested as a novel epidemiological surveillance tool to identify circulating arboviruses.