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The detection of Aedes albopictus in Lucky bamboo (Dracaena sanderiana) greenhouses and Ae. atropalpus at used tire importers illustrates that the Netherlands is exposed to the risk of introductions of invasive mosquito species (IMS). In this study we implemented a risk-based and adaptive surveillance (2010–16) in order to detect introductions and prevent potential proliferation of IMS at these locations. Results at Lucky bamboo greenhouses show that interceptions of Ae. albopictus occurred every year, with 2010 and 2012 being the years with most locations found positive for this species (n = 6), and 2015 the year with the highest percentage of positive samples (4.1%). Furthermore, our results demonstrate that Ae. japonicus can also be associated with the import of Lucky bamboo. At used tire companies, IMS were found at 12 locations. Invasive mosquito species identified were Ae. albopictus, Ae. atropalpus, Ae. aegypti, and Ae. japonicus, of which Ae. albopictus has been found every year since 2010. The proportion of samples containing IMS was significantly higher before application of a covenant between the used tire importers and the Dutch government in 2013 (12.96%) than in the successive 3 years (2014 [6.93%], 2015 [4.24%], 2016 [5.09%], 1-sided binomial test, P < 0.01). It is concluded that risk-based and adaptive surveillance is an effective methodology for detection of IMS, and that application of governmental management measures in combination with mosquito control has stabilized the situation.

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.

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.

Additional file 1. Mosquito species collected using CDC light traps in selected villages in Busoro and Ruhuha sector, Rwanda (2017)

Additional file 2. Mosquito species collected using CDC light traps in selected villages in Busoro and Ruhuha sector, Rwanda (2018)

Additional file 3. Members of the Anopheles gambiae complex found among samples of Anopheles gambiae s.l. tested from Ruhuha and Busoro sector, Ruhuha

BackgroundMalaria remains a major public health concern in the Democratic Republic of the Congo (DRC) and its control is affected by recurrent conflicts. Médecins Sans Frontières (MSF) initiated several studies to better understand the unprecedented incidence of malaria to effectively target and implement interventions in emergency settings. The current study evaluated the main vector species involved in malaria transmission and their resistance to insecticides, with the aim to propose the most effective tools and strategies for control of local malaria vectors.MethodsThis study was performed in 52 households in Shamwana (Katanga, 2014), 168 households in Baraka (South Kivu, 2015) and 269 households in Kashuga (North Kivu, 2017).Anophelesvectors were collected and subjected to standardized Word Health Organization (WHO) and Center for Disease Control (CDC) insecticide susceptibility bioassays. Mosquito species determination was done using PCR andPlasmodium falciparuminfection in mosquitoes was assessed by ELISA targeting circumsporozoite protein.ResultsOf 3517Anophelesspp. mosquitoes collected,Anopheles gambiaesensu lato (s.l.) (29.6%) andAnopheles funestus(69.1%) were the main malaria vectors. Plasmodium falciparuminfection rates forAn. gambiaes.l. were 1.0, 2.1 and 13.9% for Shamwana, Baraka and Kashuga, respectively.Anopheles funestusshowed positivity rates of 1.6% in Shamwana and 4.4% in Baraka. NoAn. funestuswere collected in Kashuga. Insecticide susceptibility tests showed resistance development towards pyrethroids in all locations. Exposure to bendiocarb, malathion and pirimiphos-methyl still resulted in high mosquito mortality.ConclusionsThis is one of only few studies from these conflict areas in DRC to report insecticide resistance in local malaria vectors. The data suggest that current malaria prevention methods in these populations are only partially effective, and require additional tools and strategies. Importantly, the results triggered MSF to consider the selection of a new insecticide for indoor residual spraying (IRS) and a new long-lasting insecticide-treated net (LLIN). The reinforcement of correct usage of LLINs and the introduction of targeted larviciding were also included as additional vector control tools as a result of the studies.

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.

BackgroundMalaria 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.MethodsThis 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.ResultsMalaria-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.ConclusionThe 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.

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-Barr&eacute, 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&ndash, 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&ndash, 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.

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.

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.

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.

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

The oviposition behavior of mosquitoes is mediated by chemical cues. In the malaria mosquito Anopheles gambiae, conspecific larvae produce infochemicals that affect this behavior. Emanations from first instar larvae proved strongly attractive to gravid females, while those from fourth instars caused oviposition deterrence, suggesting that larval developmental stage affected the oviposition choice of the female mosquito. We examined the nature of these chemicals by headspace collection of emanations of water in which larvae of different stages were developing. Four chemicals with putative effects on oviposition behavior were identified: dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) were identified in emanations from water containing fourth instars; nonane and 2,4-pentanedione (2,4-PD) were identified in emanations from water containing both first and fourth instars. Dual-choice oviposition studies with these compounds were done in the laboratory and in semi-field experiments in Tanzania. In the laboratory, DMDS and DMTS were associated with oviposition-deterrent effects, while results with nonane and 2,4-PD were inconclusive. In further studies DMDS and DMTS evoked egg retention, while with nonane and 2,4-PD 88% and 100% of female mosquitoes, respectively, laid eggs. In dual-choice semi-field trials DMDS and DMTS caused oviposition deterrence, while nonane and 2,4-PD evoked attraction, inducing females to lay more eggs in bowls containing these compounds compared to the controls. We conclude that oviposition of An. gambiae is mediated by these four infochemicals associated with conspecific larvae, eliciting either attraction or deterrence. High levels of egg retention occurred when females were exposed to chemicals associated with fourth instar larvae. Electronic supplementary material The online version of this article (10.1007/s10886-020-01175-5) contains supplementary material, which is available to authorized users.

Tandem repeat elements such as the highly diverse class of satellite repeats occupy large parts of eukaryotic chromosomes. Most occur at (peri)centromeric and (sub)telomeric regions and have been implicated in chromosome organization, stabilization, and segregation1. Others are located more dispersed throughout the genome, but their functions remained largely enigmatic. Satellite repeats in euchromatic regions were hypothesized to regulate gene expression in cis by modulation of the local heterochromatin, or in trans via repeat-derived transcripts2,3. Yet, due to a lack of experimental models, gene regulatory potential of satellite repeats remains largely unexplored. Here we show that, in the vector mosquito Aedes aegypti, a satellite repeat promotes sequence-specific gene silencing via the expression of two abundant PIWI-interacting RNAs (piRNAs). Strikingly, whereas satellite repeats and piRNA sequences generally evolve extremely fast4-6, this locus was conserved for approximately 200 million years, suggesting a central function in mosquito biology. Tandem repeat-derived piRNA production commenced shortly after egg-laying and inactivation of the most abundant of the two piRNAs in early embryos resulted in an arrest of embryonic development. Transcriptional profiling in these embryos revealed the failure to degrade maternally provided transcripts that are normally cleared during maternal-to-zygotic transition. Our results reveal a novel mechanism in which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing, which is fundamental to embryonic development. These findings highlight the regulatory potential of this enigmatic class of repeats.

Additional file 3: Table S3. A COREQ checklist highlighting details of methods.

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. © 2020 European Centre for Disease Prevention and Control (ECDC). All rights reserved.

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., Dutch Ministry of Health, Welfare and Sport (VWS); European Interreg North Sea Region program as part of the NorthTick project, This study was funded by the Dutch Ministry of Health, Welfare and Sport (VWS), and by a grant from the European Interreg North Sea Region program, as part of the NorthTick project.

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 P = 0.729). In the field, we captured Ae. aegypti, Cx. quinquefasciatus and Cx. nigripalpus. We confirmed the laboratory results and found that the combination of the MB5 blend and CO2 almost doubled Ae. aegypti female captures (P = 0.004) and more than doubled Culex spp. female captures (P = 0.005) compared to using only CO2. Interestingly, the MB5 blend outperformed the commercially available BG-Lure, in the BG-Sentinel (P Ae. aegypti when baited with the MB5 blend in similar numbers as the BG-Sentinel baited with the MB5 (P = 0.362). Conclusions Our study demonstrated that the BG-Sentinel trap baited with the MB5 blend and CO2 outperforms the current golden standard (BG-Sentinel trap with BG-Lure) for monitoring Ae. aegypti females and males, in both laboratory and field experiments. The BG-Bowl baited with the MB5 blend is a good candidate for home use. Finally, the results show that CO2 is an indispensable component of the attractive blend.

Additional file 1: Table S1. Interview guide (FGD) for Community members, HAs and LSM committees.

Additional file 1: Figure S1. Presence records and species distribution map obtained with Aedes albopictus Maxent model. Figure S2. Relationship between nineteen European bioclimatic variables (http://www.worldclim.com) for occurrence records buffers of 200 km of radius with histogram and Kernel density (diagonal), Pearson’s correlation coefficient (r) and its significance (above the diagonal) and scatterplot and linear regression (below the diagonal). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure S3. Cluster with nineteen European bioclimatic variables (http://www.worldclim.com) for occurrence records buffers of 200 km of radius. Figure S4. Map with eleven presence records of Aedes albopictus in the Netherlands. The sites are Almere, Assen, Emmeloord, Etten-Leur, Hardenberg, Lelystad, Moerdijk, Montfoort, Oosterhout, Oss, Weert. Map by authors. Figure S5. Maps showing maximum, median, minimum and standard deviation of habitat suitability obtained with Aedes albopictus Maxent model. Figure S6. The relative contribution of five environment variables (BIO11, BIO8, BIO7, BIO2, BIO12) to the model according to the regularized training gain of jackknife test in the Aedes albopictus Maxent model. Blue bars show the influence of each variable alone and green bars show the performance of the model when the variable is removed from the model. Figure S7. Response curves showing the relationships between the probability of presence of Aedes albopictus and five environment variables (BIO11, BIO8, BIO7, BIO2, BIO12). Figure S8. A scatter plot and the corresponding regression line and regression equation for the relationship between the dependent variable air temperature TG (°C × 0.1) and the independent variable LST (°C × 0.01). Abbreviations: r, Pearson’s correlation coefficient; R-square linear, coefficient of determination; P, P-value.

Background The Asian bush mosquito Aedes japonicus is invading Europe and was first discovered in Lelystad, the Netherlands in 2013, where it has established a permanent population. In this study, we investigated the vector competence of Ae. japonicus from the Netherlands for the emerging Zika virus (ZIKV) and zoonotic Usutu virus (USUV). ZIKV causes severe congenital microcephaly and Guillain-Barré syndrome in humans. USUV is closely related to West Nile virus, has recently spread throughout Europe and is causing mass mortality of birds. USUV infection in humans can result in clinical manifestations ranging from mild disease to severe neurological impairments. Methodology/Principal findings In our study, field-collected Ae. japonicus females received an infectious blood meal with ZIKV or USUV by droplet feeding. After 14 days at 28°C, 3% of the ZIKV-blood fed mosquitoes and 13% of the USUV-blood fed mosquitoes showed virus-positive saliva, indicating that Ae. japonicus can transmit both viruses. To investigate the effect of the mosquito midgut barrier on virus transmission, female mosquitoes were intrathoracically injected with ZIKV or USUV. Of the injected mosquitoes, 96% (ZIKV) and 88% (USUV) showed virus-positive saliva after 14 days at 28°C. This indicates that ZIKV and USUV can efficiently replicate in Ae. japonicus but that a strong midgut barrier is normally restricting virus dissemination. Small RNA deep sequencing of orally infected mosquitoes confirmed active replication of ZIKV and USUV, as demonstrated by potent small interfering RNA responses against both viruses. Additionally, de novo small RNA assembly revealed the presence of a novel narnavirus in Ae. japonicus. Conclusions/Significance Given that Ae. japonicus can experimentally transmit arthropod-borne viruses (arboviruses) like ZIKV and USUV and is currently expanding its territories, we should consider this mosquito as a potential vector for arboviral diseases in Europe., Author summary Arthropod-borne viruses (arboviruses) cause a high disease burden in humans and animals. Zika virus (ZIKV) causes microcephaly and Guillain-Barré syndrome in humans, whereas Usutu virus (USUV) induces high mortality in birds and neurological disease in humans. The spread of arboviruses such as ZIKV and USUV is determined by the presence of mosquitoes that can transmit these viruses from one vertebrate host to the next. Here, we investigate the risk of transmission of ZIKV and USUV by the Asian bush mosquito Aedes japonicus. This mosquito is invading Europe and is currently present in the Netherlands. We found that field-collected Ae. japonicus mosquitoes can experimentally transmit ZIKV and USUV. Of the orally infected mosquitoes, 3% (ZIKV) and 13% (USUV) showed virus-positive saliva after 14 days at 28°C. We also found that ZIKV and USUV activated the antiviral RNA interference immune response of Ae. japonicus. Moreover, a strong barrier in the mosquito midgut restricted virus dissemination, since 96% (ZIKV) and 88% (USUV) of the mosquitoes injected with ZIKV or USUV showed virus-positive saliva. Additionally, we discovered a narnavirus in Ae. japonicus. Given that Ae. japonicus can transmit ZIKV and USUV, we should consider this mosquito as a potential vector for arboviral diseases in Europe.

The decay kinetics of the redox states S2 and S3 of the water-oxidizing enzyme have been analyzed in isolated spinach thylakoids in the absence and presence of the exogenous reductant hydrazine. In control samples without NH2NH2 a biphasic decay is observed. The rapid decline of S2 and S3 with YD as reductant exhibits practically the same kinetics with t1/2 = 6-7 s at pH = 7.2 and 7 degrees C. The slow reduction (order of 5-10 min at 7 degrees C) of S2 and S3 with endogenous electron donors other than YD is about twice as fast for S2 as for S3 under these conditions. In contrast, the hydrazine-induced reductive shifts of the formal redox states Si (i = 0...3) are characterized by a totally different kinetic pattern: (a) at 1 mM NH2NH2 and incubation on ice the decay of S2 is estimated to be at least 25 times faster (t1/2 less than or equal to 0.4 min) than the corresponding reaction of S3 (t1/2 approximately 13 min); (b) the NH2NH2-induced decay of S3 is even slower (about twice) than the transformation of S1 into the formal redox state 'S-1' (t1/2 approximately 6 min), which gives rise to the two-digit phase shift of the oxygen-yield pattern induced by a flash train in dark adapted thylakoids. (c) the NH2NH2-induced transformation S0----'S-2' [Renger, Messinger and Hanssum (1990) in: Curr.' Res. Photosynth. (Baltscheffsky, M., ed), Vol. 1, pp. 845-848, Kluwer, Dordrecht] is about three times faster (t1/2 approximately 2 min) than the reaction [see text]. Based on these results, the following dependence on the redox state Si of the reactivity towards NH2NH2 is obtained: S3 less than S1 less than S0 much less than S2. The implications of this surprising order of reactivity are discussed.

Tripartite interactions among insect vectors, midgut bacteria, and viruses may determine the ability of insects to transmit pathogenic arboviruses. Here, we investigated the impact of gut bacteria on the susceptibility of Culicoides nubeculosus and Culicoides sonorensis biting midges for Schmallenberg virus, and of Aedes aegypti mosquitoes for Zika and chikungunya viruses. Gut bacteria were manipulated by treating the adult insects with antibiotics. The gut bacterial communities were investigated using Illumina MiSeq sequencing of 16S rRNA, and susceptibility to arbovirus infection was tested by feeding insects with an infectious blood meal. Antibiotic treatment led to changes in gut bacteria for all insects. Interestingly, the gut bacterial composition of untreated Ae. aegypti and C. nubeculosus showed Asaia as the dominant genus, which was drastically reduced after antibiotic treatment. Furthermore, antibiotic treatment resulted in relatively more Delftia bacteria in both biting midge species, but not in mosquitoes. Antibiotic treatment and subsequent changes in gut bacterial communities were associated with a significant, 1.8-fold increased infection rate of C. nubeculosus with Schmallenberg virus, but not for C. sonorensis. We did not find any changes in infection rates for Ae. aegypti mosquitoes with Zika or chikungunya virus. We conclude that resident gut bacteria may dampen arbovirus transmission in biting midges, but not so in mosquitoes. Use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals, by increasing the transmission of viral pathogens by biting midges.

Additional file 1. Additional tables.

Despite the implementation of a number of interventions aimed at controlling malaria, Rwanda is experiencing a countrywide resurgence of simple malaria cases over the past five years. To support malaria control, mosquito surveillance activities, such as systematic reporting of the distribution, the diversity and the infectivity rate of malaria vectors throughout the country, have been undertaken. However, mosquito monitoring programmes are not carried out to monitor the impact of all vector control interventions or to determine the distribution of mosquito species in all areas, especially in the remote regions of the country. With a target of reducing malaria mortality by 2020, implementation of mosquito surveillance in those regions is urgently needed as well. In this paper, a Citizen science approach as a capacity resource for malaria vector monitoring for the Rwandan National Malaria Control Programme is presented. The ultimate aim is to complement existing mosquito surveillance currently in place by providing key information on the spatio-temporal distribution of mosquito nuisance and malaria vectors. This will contribute to an insight into the ecology of malaria vectors and thereby to a better understanding of malaria transmission patterns in Rwanda.

Malaria remains a major threat to public health. Long-Lasting Insecticide-treated Nets (LLINs) and Indoor Residual Spraying (IRS) have been widely adopted as important malaria prevention and control interventions and have contributed to significant reduction in malaria incidence. However, recently malaria resurgence has been reported in different countries, including Rwanda, indicating that current attempts to control and eliminate malaria may be failing due to environmental changes and changes in human behaviour. Engaging citizens in malaria prevention and control would help them to identify and prioritize their own health concerns and be able to make appropriate decisions. A citizen science approach to monitor ecological changes by providing timely information is likely to support more effective and consistent use of malaria prevention and control interventions. However, the application of citizen science in malaria prevention and control has lagged behind when comparing with areas of disease diagnosis and treatment. In addition, the determinants of participation in such a citizen science program have not been fully analyzed. This paper presents a conceptual model of likely determinants of participation in connective action (sharing and exchange of malaria-related information), effective and consistent use of malaria preventive and control measures (LLINs and IRS) and collective action (participating in public goods for malaria prevention). The model will guide future research on behavioural and contextual factors and may enhance the effective and consistent use of malaria preventive and control interventions.

To date, the Republic of Rwanda has not systematically reported on distribution, diversity and malaria infectivity rate of mosquito species throughout the country. Therefore, we assessed the spatial and temporal variation of mosquitoes in the domestic environment, as well as the nocturnal biting behavior and infection patterns of the main malaria vectors in Rwanda. For this purpose, mosquitoes were collected monthly from 2010 to 2013 by human landing catches (HLC) and pyrethrum spray collections (PSC) in seven sentinel sites. Mosquitoes were identified using morphological characteristics and PCR. Plasmodium falciparum sporozoite infection rates were determined using ELISA. A total of 340,684 mosquitoes was collected by HLC and 73.8% were morphologically identified as culicines and 26.2% as anophelines. Of the latter, 94.3% were Anopheles gambiae s.l., 0.4% Anopheles funestus and 5.3% other Anopheles species. Of An. gambiae s.l., An. arabiensis and An. gambiae s.s. represented 84.4% and 15.6%, respectively. Of all An. gambiae s.l. collected indoor and outdoor, the proportion collected indoors was 51.3% in 2010 and 44.9% in 2013. A total of 17,022 mosquitoes was collected by PSC of which 20.5% were An. gambiae s.l. and 79.5% were culicines. For the seven sentinel sites, the mean indoor density for An. gambiae s.l. varied from 0.0 to 1.0 mosquitoes/house/night. P. falciparum infection rates in mosquitoes varied from 0.87 to 4.06%. The entomological inoculation rate (EIR) ranged from 1.0 to 329.8 with an annual average of 99.5 infective bites/person/year. This longitudinal study shows, for the first time, the abundance, species composition, and entomological inoculation rate of malaria mosquitoes collected throughout Rwanda.

Isoxazolines are oral insecticidal drugs currently licensed for ectoparasite control in companion animals. Here we propose their use in humans for the reduction of vector-borne disease incidence. Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and Culex mosquitoes and Phlebotomus sand flies after feeding on a drug-supplemented blood meal, with IC50 values ranging from 33 to 575 nM, and were fully active against strains with preexisting resistance to common insecticides. Based on allometric scaling of preclinical pharmacokinetics data, we predict that a single human median dose of 260 mg (IQR, 177-407 mg) for afoxolaner, or 410 mg (IQR, 278-648 mg) for fluralaner, could provide an insecticidal effect lasting 50-90 days against mosquitoes and Phlebotomus sand flies. Computational modeling showed that seasonal mass drug administration of such a single dose to a fraction of a regional population would dramatically reduce clinical cases of Zika and malaria in endemic settings. Isoxazolines therefore represent a promising new component of drug-based vector control.

Este artículo contiene 18 páginas, 2 tablas, 2 figuras., Public involvement in science has allowed researchers to collect large-scale and real-time data and also engage citizens, so researchers are adopting citizen science (CS) in many areas. One promising appeal is student participation in CS school programs. In this literature review, we aimed to investigate which school CS programs exist in the areas of (applied) life sciences and if any projects target infectious disease surveillance. This review’s objectives are to determine success factors in terms of data quality and student engagement. After a comprehensive search in biomedical and social databases, we found 23 projects. None of the projects found focused on infectious disease surveillance, and the majority centered around species biodiversity. While a few projects had issues with data quality, simplifying the protocol or allowing students to resubmit data made the data collected more usable. Overall, students at different educational levels and disciplines were able to collect usable data that was comparable to expert data and had positive learning experiences. In this review, we have identified limitations and gaps in reported CS school projects and provided recommendations for establishing future programs. This review shows the value of using CS in collaboration with traditional research techniques to advance future science and increasingly engage communities., This work is part of the research programme One Health PACT with project number 109986, which is (partly) financed by the Dutch Research Council (NWO).

In Europe, West Nile virus (WNV) outbreaks have been limited to southern and central European countries. However, competent mosquito vectors and susceptible bird hosts are present in northern Europe. Differences in temperature and vector competence of mosquito populations may explain the absence of WNV outbreaks in northern Europe. The aim of the present study was to directly compare vector competence of northern and southern European Culex pipiens (Cx. p.) pipiens mosquitoes for WNV across a gradient of temperatures. WNV infection and transmission rates were determined for two Cx. p. pipiens populations originating from The Netherlands and Italy, respectively. Mosquitoes were orally exposed by providing an infectious bloodmeal, or by injecting WNV (lineage 2) in the thorax, followed by 14-day incubation at 18, 23, or 28 °C. No differences in infection or transmission rates were found between the Cx. p. pipiens populations with both infection methods, but WNV transmission rates were significantly higher at temperatures above 18 °C. The absence of WNV outbreaks in northern Europe cannot be explained by differences in vector competence between Cx. p. pipiens populations originating from northern and southern Europe. This study suggests that low temperature is a key limiting factor for WNV transmission.

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&trade, 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.

A promising strategy for reducing the transmission of dengue and other arboviral human diseases by Aedes aegypti mosquito vector populations involves field introductions of the endosymbiotic bacteria Wolbachia. Wolbachia infections inhibit viral transmission by the mosquito, and can spread between mosquito hosts to reach high frequencies in the vector population. Wolbachia spreads by maternal transmission, and spread dynamics can be variable and highly dependent on natural mosquito population dynamics, population structure and fitness components. We develop a mathematical model of an A. aegypti metapopulation that incorporates empirically validated relationships describing density-dependent mosquito fitness components. We assume that density dependent relationships differ across subpopulations, and construct heterogeneous landscapes for which model-predicted patterns of variation in mosquito abundance and demography approximate those observed in field populations. We then simulate Wolbachia release strategies similar to that used in field trials. We show that our model can produce rates of spatial spread of Wolbachia similar to those observed following field releases. We then investigate how different types of spatio-temporal variation in mosquito habitat, as well as different fitness costs incurred by Wolbachia on the mosquito host, influence predicted spread rates. We find that fitness costs reduce spread rates more strongly when the habitat landscape varies temporally due to stochastic and seasonal processes. Synthesis and applications: Our empirically based modelling approach represents effects of environmental heterogeneity on the spatial spread of Wolbachia. The models can assist in interpreting observed spread patterns following field releases and in designing suitable release strategies for targeting spatially heterogeneous vector populations.

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.

Good health and human wellbeing is one of the sustainable development goals. To achieve this goal, many efforts are required to control infectious diseases including malaria which remains a major public health concern in Rwanda. Surveillance of mosquitoes is critical to control the disease, but surveillance rarely includes the participation of citizens. A citizen science approach (CSA) has been applied for mosquito surveillance in developed countries, but it is unknown whether it is feasible in rural African contexts. In this paper, the technical and social components of such a program are described. Participatory design workshops were conducted in Ruhuha, Rwanda. Community members can decide on the technical tools for collecting and reporting mosquito species, mosquito nuisance, and confirmed malaria cases. Community members set up a social structure to gather observations by nominating representatives to collect the reports and send them to the researchers. These results demonstrate that co-designing a citizen science program (CSP) with citizens allows for decision on what to use in reporting observations. The decisions that the citizens took demonstrated that they have context-specific knowledge and skills, and showed that implementing a CSP in a rural area is feasible.

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.

Background Shuni virus (SHUV) is an orthobunyavirus that belongs to the Simbu serogroup. SHUV was isolated from diverse species of domesticated animals and wildlife, and is associated with neurological disease, abortions, and congenital malformations. Recently, SHUV caused outbreaks among ruminants in Israel, representing the first incursions outside the African continent. The isolation of SHUV from a febrile child in Nigeria and seroprevalence among veterinarians in South Africa suggests that the virus may have zoonotic potential as well. The high pathogenicity, extremely broad tropism, potential transmission via both biting midges and mosquitoes, and zoonotic features warrants prioritization of SHUV for further research. Additional knowledge is essential to accurately determine the risk for animal and human health, and to assess the risk of future epizootics and epidemics. To gain first insights into the potential involvement of arthropod vectors in SHUV transmission, we have investigated the ability of SHUV to infect and disseminate in laboratory-reared biting midges and mosquitoes. Methodology/Principal findings Culicoides nubeculosus, C. sonorensis, Culex pipiens pipiens, and Aedes aegypti were orally exposed to SHUV by providing an infectious blood meal. Biting midges showed high infection rates of approximately 40–60%, whereas infection rates of mosquitoes were lower than 2%. SHUV successfully disseminated in both species of biting midges, but no evidence of transmission in orally exposed mosquitoes was found. Conclusions/Significance The results of this study show that different species of Culicoides biting midges are susceptible to infection and dissemination of SHUV, whereas the two mosquito species tested were found not to be susceptible., Author summary Arthropod-borne (arbo)viruses are notorious for causing unpredictable and large-scale epidemics and epizootics. Apart from viruses such as West Nile virus and Rift Valley fever virus that are well known to have a significant impact on human and animal health, many arboviruses remain neglected. Shuni virus (SHUV) is a neglected virus with zoonotic potential that was recently associated with severe disease in livestock and wildlife. Isolations of SHUV from field-collected biting midges and mosquitoes suggests that SHUV may be transmitted by these insects. Laboratory-reared biting midge species (Culicoides nubeculosus and C. sonorensis) and mosquito species (Culex pipiens pipiens and Aedes aegypti), that are known to transmit other arboviruses, were exposed to SHUV via an infectious blood meal. SHUV was able to successfully disseminate in both biting midge species, whereas no evidence of infection or transmission in both mosquito species was found. Our results show that SHUV infects and disseminates in two different Culicoides species, suggesting that these insects could play an important role in the disease transmission cycle.

Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG dinucleotide frequencies showed substantially enhanced replication compared to wild type. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG- or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wild-type virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on dinucleotide composition. However, the evolutionary pressure on the CpG dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arbo flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector., The genome of the flavivirus Zika virus is stuck in a tug-of-war between two directly opposing evolutionary pressures that are present in the cells of mammalian host organisms and mosquito vectors; this results in an adaptive compromise, as manifested in the virus’s genome dinucleotide composition.

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5′-3′ exoribonuclease XRN1/Pacman on conserved RNA structures in the 3′ untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo . Two reproducible small-RNA hot spots within the 3′ UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3′ SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

Introduction: Increasing the knowledgebase of anopheline larval ecology could enable targeted deployment of malaria control efforts and consequently reduce costs of implementation. In Malawi, there exists a knowledge gap in anopheline larval ecology and, therefore, basis for targeted deployment of larval source management (LSM) for malaria control, specifically larvicides. We set out to characterize anopheline larval habitats in the Majete area of Malawi on the basis of habitat ecology and anopheline larval productivity to create a basis for larval control initiatives in the country. Methods: Longitudinal surveys were conducted in randomly selected larval habitats over a period of fifteen months in Chikwawa district, southern Malawi. Biotic and abiotic parameters of the habitats were modelled to determine their effect on the occurrence and densities of anopheline larvae. Results: Seventy aquatic habitats were individually visited between 1-7 times over the study period. A total of 5,123 immature mosquitoes (3,359 anophelines, 1,497 culicines and 267 pupae) were collected. Anopheline and culicine larvae were observed in sympatry in aquatic habitats. Of the nine habitat types followed, dams, swamps, ponds, borehole runoffs and drainage channels were the five most productive habitat types for anopheline mosquitoes. Anopheline densities were higher in aquatic habitats with bare soil making up part of the surrounding land cover (p

A promising strategy for reducing the transmission of dengue and other arboviral human diseases byAedes aegyptimosquito vector populations involves field introductions of the endosymbiotic bacteriaWolbachia.Wolbachiainfections inhibit viral transmission by the mosquito, and can spread between mosquito hosts to reach high frequencies in the vector population.Wolbachiaspreads by maternal transmission, and spread dynamics can be variable and highly dependent on natural mosquito population dynamics, population structure and fitness components.We develop a mathematical model of anAe. aegyptimetapopulation that incorporates empirically validated relationships describing density-dependent mosquito fitness components. We assume that density dependence relationships differ across subpopulations, and construct heterogeneous landscapes for which model-predicted patterns of variation in mosquito abundance and demography approximate those observed in field populations. We then simulateWolbachiarelease strategies similar to that used in field trials.We show that our model can produce rates of spatial spread ofWolbachiasimilar to those observed following field releases.We then investigate how different types of spatio-temporal variation in mosquito habitat, as well as different fitness costs incurred byWolbachiaon the mosquito host, influence predicted spread rates. We find that fitness costs reduce spread rates more strongly when the habitat landscape varies temporally due to stochastic and seasonal processes.Our empirically based modelling approach represents effects of environmental heterogeneity on the spatial spread ofWolbachia.The models can assist in interpreting observed spread patterns following field releases and in designing suitable release strategies for targeting spatially heterogeneous vector populations.

To sustain reductions in child mortality due to malaria, J. Hemingway argues that new public health insecticides are required (“The way forward for vector control,” Perspectives, 24 November 2017, p. [998][1]). However, because of the heavy selection pressure placed on mosquito populations, the

Background Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear. Methodology / Principal findings Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti. Conclusions / Significance The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies., Author summary Chikungunya virus (CHIKV) is a re-emerging arthropod-borne virus that is transmitted predominantly by Aedes aegypti mosquitoes. In 2016 alone CHIKV caused over 100.000 infections in South-America, exemplifying the impact of CHIKV disease. Previous research has suggested that the CHIKV non-structural protein 3 (nsP3) may determine the infection of mosquitoes. NsP3 is known to interact with several host proteins through a conserved proline (P)-rich and duplicate FGDF motifs that are present in its C-terminal domain. Here we investigated the importance of these conserved motifs for the infection and replication of CHIKV in both Aedes mosquito cells and mammalian cells. Furthermore, we assessed the role of these motifs for the transmission by Ae. aegypti mosquitoes via infectious bloodmeal experiments. We show that mutation of the P-rich motif negatively affects the replication of CHIKV in both mammalian and mosquito cells. In contrast, mutating the P-rich motif did not affect the transmission by Ae. aegypti. Mutation of both FGDF motifs together completely inactivated CHIKV in mammalian and mosquito cells, while mutation of a single FGDF motif negatively affected replication only in mammalian cells. Importantly, CHIKV containing only a single FGDF motif was still efficiently transmitted by Ae. aegypti mosquitoes. These results contribute to understanding the key interactions between alphaviruses and their mosquito vector.

Vector-borne animal diseases pose a continuous and substantial threat to livestock economies around the globe. Increasing international travel, the globalisation of trade, and climate change are likely to play a progressively more important role in the introduction, establishment and spread of arthropod-borne pathogens worldwide. A review of the literature reveals that many climatic variables, functioning singly or in combination, exert varying effects on the distribution and range of Culicoides vector midges and mosquitoes. For example, higher temperatures may be associated with increased insect abundance--thereby amplifying the risk of disease transmission--but there are no indications yet of dramatic shifts occurring in the geographic range of Culicoides midges. However, the same cannot be said for mosquitoes: over the last few decades, multiple Asian species have established themselves in Europe, spread and are unlikely to ever be eradicated. Research on how insects respond to changes in climate is still in its infancy. The authors argue that we need to grasp how other annectant changes, such as extremes in precipitation (drought and flooding), may affect the dispersal capability of mosquitoes. Models are useful for assessing the interplay between mosquito vectors expanding their range and the native flora and fauna; however, ecological studies employing classical mark-release-recapture techniques remain essential for addressing fundamental questions about the survival and dispersal of mosquito species, with the resulting parameters fed directly into new-generation disease transmission models. Studies on the eventual impact of mosquitoes on animal and human health should be tackled through large-scale integrated research programmes. Such an approach calls for more collaborative efforts, along the lines of the One Health Initiative.

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. Funding Agencies|Ministry of Economic Affairs, the Netherlands [1300018161]; Swedish Research Council (FORMAS), Sweden; Italian Ministry of Education, University and Research (MIUR) [2012T85B3R]

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 (R0) models. However, existing R0 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 R0 model with temperature-dependent and biotype-specific parameters, and calculated R0 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 R0. 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 R0 shows the importance of including biotype-specific parameters in models for reliable WNV risk assessments.