A satellite repeat-derived piRNA controls embryonic development of Aedes.

Tandem repeat elements such as the diverse class of satellite repeats occupy large parts of eukaryotic chromosomes, mostly at centromeric, pericentromeric, telomeric and subtelomeric regions1. However, some elements are located in euchromatic regions throughout the genome and have been hypothesized to regulate gene expression in cis by modulating local chromatin structure, or in trans via transcripts derived from the repeats2-4. Here we show that a satellite repeat in the mosquito Aedes aegypti promotes sequence-specific gene silencing via the expression of two PIWI-interacting RNAs (piRNAs). Whereas satellite repeats and piRNA sequences generally evolve extremely quickly5-7, this locus was conserved for approximately 200 million years, suggesting that it has a central function in mosquito biology. piRNA production commenced shortly after egg laying, and inactivation of the more abundant piRNA resulted in failure to degrade maternally deposited transcripts in the zygote and developmental arrest. Our results reveal a mechanism by which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing that is essential for embryonic development.

Incomplete bunyavirus particles can cooperatively support virus infection and spread.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors.

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.

Fermenting molasses and a synthetic odour blend to attract blood-fed Anopheles coluzzii.

Collecting blood-fed mosquitoes to monitor pathogen presence or to gather information on the host blood meal is often challenging. Fermenting molasses can be used to produce carbon dioxide to attract host-seeking mosquitoes, however, earlier work indicated that it may also attract blood-fed mosquitoes in the field. In the current study, these field results were validated in an experimental setting using a large cage setup with Anopheles coluzzii (Diptera, Culicidae). Blood-fed mosquitoes were indeed attracted to fermenting molasses with the highest attraction at 72 hours post feeding, which was used for subsequent experiments. Next, it was tested if fermentation of molasses is required for attraction, and whether it acts as an oviposition attractant, increases egg laying, or increases mosquito survival. The compounds that could be responsible for attraction were identified by combined electrophysiology and chemical analyses and formulated into a synthetic blend. Fermenting molasses attracted blood-fed mosquitoes in the large cage study, while fermenting sugar and non-fermenting molasses did not. The fecundity of blood-fed mosquitoes increased after feeding on fermenting molasses, however, compounds emanating from molasses did not trigger oviposition. The synthetic blend attracted blood-fed mosquitoes and may be used to determine mosquito host selection and for xenomonitoring, as 'flying syringes' to detect non-vector borne pathogens.

Continued Circulation of Tick-Borne Encephalitis Virus Variants and Detection of Novel Transmission Foci, the Netherlands.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen that was first detected in ticks and humans in the Netherlands in 2015 (ticks) and 2016 (humans). To learn more about its distribution and prevalence in the Netherlands, we conducted large-scale surveillance in ticks and rodents during August 2018-September 2020. We tested 320 wild rodents and >46,000 ticks from 48 locations considered to be at high risk for TBEV circulation. We found TBEV RNA in 3 rodents (0.9%) and 7 tick pools (minimum infection rate 0.02%) from 5 geographically distinct foci. Phylogenetic analyses indicated that 3 different variants of the TBEV-Eu subtype circulate in the Netherlands, suggesting multiple independent introductions. Combined with recent human cases outside known TBEV hotspots, our data demonstrate that the distribution of TBEV in the Netherlands is more widespread than previously thought.

Species Identity, Life History, and Geographic Distance Influence Gut Bacterial Communities in Lab-Reared and European Field-Collected Culicoides Biting midges.

Bacteria are part of the insect gut system and influence many physiological traits of their host. Gut bacteria may even reduce or block the transmission of arboviruses in several species of arthropod vectors. Culicoides biting midges are important arboviral vectors of several livestock and wildlife diseases, yet limited information is available on their gut bacterial communities. Addressing this gap will help inform how these communities can be manipulated and ultimately used as novel tools to control pathogens. To assess how bacterial communities change during the life stages of lab-reared C. nubeculosus and C. sonorensis, endosymbiotic bacteria were identified using Illumina sequencing of 16S rRNA and taxonomically characterised. Analyses were conducted to determine how gut bacterial communities in adults are influenced by species identity and geographic distance among biting midge populations. Communities of the two lab-reared Culicoides species significantly changed after pupation and with maturation into 6-day-old adults. Pseudomonas, Burkholderiaceae and Leucobacter bacteria were part of a core community that was trans-stadially transmitted and found throughout their life cycle. Among field-collected biting midges, the bacterial communities were unique for almost each species. Cardinium, Rickettsia and Wolbachia were some of the most abundant bacteria in midges collected from wetlands. Only Pseudomonas was present in high relative abundance in all field-collected species. In this study, species identity, as well as geographic distance, influenced the gut bacterial communities and may partly explain known inter- and intra-species variability in vector competence. Additionally, stably associated bacterial species could be candidates for paratransgenic strategies to control vector-borne pathogens.

Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti.

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.

Monitoring mosquito nuisance for the development of a citizen science approach for malaria vector surveillance in Rwanda.

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.

Citizen science for monitoring the spatial and temporal dynamics of malaria vectors in relation to environmental risk factors in Ruhuha, Rwanda.

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.

Repurposing isoxazoline veterinary drugs for control of vector-borne human diseases.

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.

What do people benefit from a citizen science programme? Evidence from a Rwandan citizen science programme on malaria control.

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.

Community factors affecting participation in larval source management for malaria control in Chikwawa District, Southern Malawi.

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.

Identification of main malaria vectors and their insecticide resistance profile in internally displaced and indigenous communities in Eastern Democratic Republic of the Congo (DRC).

BACKGROUND: Malaria 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. METHODS: This 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). Anopheles vectors 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 and Plasmodium falciparum infection in mosquitoes was assessed by ELISA targeting circumsporozoite protein. RESULTS: Of 3517 Anopheles spp. mosquitoes collected, Anopheles gambiae sensu lato (s.l.) (29.6%) and Anopheles funestus (69.1%) were the main malaria vectors. Plasmodium falciparum infection rates for An. gambiae s.l. were 1.0, 2.1 and 13.9% for Shamwana, Baraka and Kashuga, respectively. Anopheles funestus showed positivity rates of 1.6% in Shamwana and 4.4% in Baraka. No An. funestus were 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. CONCLUSIONS: This 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.

Impact of Gut Bacteria on the Infection and Transmission of Pathogenic Arboviruses by Biting Midges and Mosquitoes.

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.

Chemical Mediation of Oviposition by Anopheles Mosquitoes: a Push-Pull System Driven by Volatiles Associated with Larval Stages.

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.

Role of individual perceptions in the consistent use of malaria preventive measures: mixed methods evidence from rural Rwanda.

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.

Virus interferes with host-seeking behaviour of mosquito.

Transmission of vector-borne pathogens is dependent on the host-seeking behaviour of their vector. Pathogen manipulation of the host-seeking behaviour of vectors towards susceptible hosts is supposedly beneficial for transmission. For West Nile virus (WNV), manipulation of the host-seeking behaviour of the main mosquito vector towards birds would be advantageous, because mammals are dead-end hosts. We hypothesised that WNV infection induces a stronger host-seeking response and a shift in host preference towards birds, to enhance its transmission by mosquitoes. However, here we show that WNV infection decreases the host-seeking response, and does not induce a shift in mosquito host preference. Other fitness-related traits are not affected by WNV infection. No effect of WNV infection was found on antennal electrophysiological responsiveness. Thus, the reduced host-seeking response is likely to result from interference in the mosquito's central nervous system. This is the first study that shows changes, specifically in the host-seeking behaviour induced by a pathogen, that do not favour transmission.

Community-based biological control of malaria mosquitoes using Bacillus thuringiensis var. israelensis (Bti) in Rwanda: community awareness, acceptance and participation.

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.

Susceptibility of Anopheles gambiae to insecticides used for malaria vector control in Rwanda.

BACKGROUND: The widespread emergence of resistance to pyrethroids is a major threat to the gains made in malaria control. To monitor the presence and possible emergence of resistance against a variety of insecticides used for malaria control in Rwanda, nationwide insecticide resistance surveys were conducted in 2011 and 2013. METHODS: Larvae of Anopheles gambiae sensu lato mosquitoes were collected in 12 sentinel sites throughout Rwanda. These were reared to adults and analysed for knock-down and mortality using WHO insecticide test papers with standard diagnostic doses of the recommended insecticides. A sub-sample of tested specimens was analysed for the presence of knockdown resistance (kdr) mutations. RESULTS: A total of 14,311 mosquitoes were tested and from a sample of 1406 specimens, 1165 (82.9%) were identified as Anopheles arabiensis and 241 (17.1%) as Anopheles gambiae sensu stricto. Mortality results indicated a significant increase in resistance to lambda-cyhalothrin from 2011 to 2013 in 83% of the sites, permethrin in 25% of the sites, deltamethrin in 25% of the sites and DDT in 50% of the sites. Mosquitoes from 83% of the sites showed full susceptibility to bendiocarb and 17% of sites were suspected to harbour resistance that requires further confirmation. No resistance was observed to fenitrothion in all study sites during the entire survey. The kdr genotype results in An. gambiae s.s. showed that 67 (50%) possessed susceptibility (SS) alleles, while 35 (26.1%) and 32 (23.9%) mosquitoes had heterozygous (RS) and homozygous (RR) alleles, respectively. Of the 591 An. arabiensis genotyped, 425 (71.9%) possessed homozygous (SS) alleles while 158 (26.7%) and 8 (1.4%) had heterozygous (RS) and homozygous (RR) alleles, respectively. Metabolic resistance involving oxidase enzymes was also detected using the synergist PBO. CONCLUSION: This is the first nationwide study of insecticide resistance in malaria vectors in Rwanda. It shows the gradual increase of insecticide resistance to pyrethroids (lambda-cyhalothrin, deltamethrin, permethrin) and organochlorines (DDT) and the large presence of target site insensitivity. The results demonstrate the need for Rwanda to expand monitoring for insecticide resistance including further metabolic resistance testing and implement an insecticide resistance management strategy to sustain the gains made in malaria control.

Using an intervention mapping approach for planning, implementing and assessing a community-led project towards malaria elimination in the Eastern Province of Rwanda.

BACKGROUND: Active community participation in malaria control is key to achieving malaria pre-elimination in Rwanda. This paper describes development, implementation and evaluation of a community-based malaria elimination project in Ruhuha sector, Bugesera district, Eastern province of Rwanda. METHODS: Guided by an intervention mapping approach, a needs assessment was conducted using household and entomological surveys and focus group interviews. Data related to behavioural, epidemiological, entomological and economical aspects were collected. Desired behavioural and environmental outcomes were identified concurrently with behavioural and environmental determinants. Theoretical methods and their practical applications were enumerated to guide programme development and implementation. An operational plan including the scope and sequence as well as programme materials was developed. Two project components were subsequently implemented following community trainings: (1) community malaria action teams (CMATs) were initiated in mid-2014 as platforms to deliver malaria preventive messages at village level, and (2) a mosquito larval source control programme using biological substances was deployed for a duration of 6 months, implemented from January to July 2015. Process and outcome evaluation has been conducted for both programme components to inform future scale up. RESULTS: The project highlighted malaria patterns in the area and underpinned behavioural and environmental factors contributing to malaria transmission. Active involvement of the community in collaboration with CMATs contributed to health literacy, particularly increasing ability to make knowledgeable decisions in regards to malaria prevention and control. A follow up survey conducted six months following the establishment of CMATs reported a reduction of presumed malaria cases at the end of 2014. The changes were related to an increase in the acceptance and use of available preventive measures, such as indoor residual spraying and increase in community-based health insurance membership, also considered as a predictor of prompt and adequate care. The innovative larval source control intervention contributed to reduction in mosquito density and nuisance bites, increased knowledge and skills for malaria control as well as programme ownership. CONCLUSION: This community-based programme demonstrated the feasibility and effectiveness of active community participation in malaria control activities, which largely contributed to community empowerment and reduction of presumed malaria in the area. Further studies should explore how gains may be sustained to achieve the goal of malaria pre-elimination.