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Female mosquitoes require blood from their host for egg development. However, the relationship between the composition of host blood and mosquito reproduction, and whether and how this is linked to host selection, remain unclear. A better understanding of these issues is beneficial for mass-rearing of mosquitoes for vector control. This review provides an overview of the currently known effects of blood constituents on mosquito reproduction. Furthermore, it highlights knowledge gaps and proposes new avenues for investigation. We recommend that research efforts be focused on physiological differences between generalist and specialist mosquito species as models to investigate if and how host preference correlates with reproductive output., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2022 Royal Entomological Society.)

Background: Evidence suggests a vicious cycle between rice cultivation and malaria control in Rwanda. Rice fields offer an attractive breeding ground for malaria vectors, which increases the disease burden in rice farming communities, and, consequently, reduces productivity in the rice sector. Community-based larval source management in rice fields is propagated as a sustainable solution to break this cycle. A sense of agency and ownership of malaria control interventions, as well as the mobilization of resources at the local level, are often considered preconditions for success. However, an evidence gap exists regarding the interaction between the agentive and financial dimension of local sustainability., Methods: We conduct a larviciding pilot involving three groups; one group where rice farmers sprayed their fields under expert supervision, one group where rice farmers organised the larviciding campaign themselves, and a (non-sprayed) control group. We test whether the difference in agency between the intervention groups affects farmers' willingness-to-pay for a larviciding campaign. Willingness-to-pay is elicited in a contingent valuation exercise, more specifically a bidding game, and is assessed both before and after the pilot (n = 288). Difference-in-difference estimates are computed, using a propensity score matching technique. Supplementary data were collected in a survey and two focus group discussions for triangulation., Results: The high-agency (self-organised) group significantly outperforms the low-agency (expert-supervised) group in terms of maintaining its willingness to contribute financially. However, higher willingness-to-pay in the high-agency group does not appear to be driven by a stronger sense of ownership per se. The supplementary data indicate high levels of ownership in both treatment groups compared to the control group. A tentative explanation lies in diverging perceptions concerning the effectiveness of the pilot., Conclusions: The study supports the idea that community-led organization of larval source management can prove instrumental in mobilizing finance for malaria control in low-income settings where rice production interferes with the fight against malaria. However, the causality is complex. Feelings of ownership do not appear the main driver of willingness-to-pay, at least not directly, which opens up the possibility of initiating community-driven malaria control interventions that promote the agentive and financial dimension of local sustainability simultaneously., (© 2023. The Author(s).)

Larval source management (LSM) could reduce malaria transmission when executed alongside core vector control strategies. Involving communities in LSM could increase intervention coverage, reduce operational costs, and promote sustainability via community buy-in. We assessed the effectiveness of community-led LSM to reduce anopheline larval densities in 26 villages along the perimeter of Majete Wildlife Reserve in southern Malawi. The communities formed LSM committees which coordinated LSM activities in their villages following specialized training. Effectiveness of larviciding by LSM committees was assessed via pre- and post-spray larval sampling. The effect of community-led LSM on anopheline larval densities in intervention villages was assessed via comparisons with densities in non-LSM villages over a period of 14 months. Surveys involving 502 respondents were undertaken in intervention villages to explore community motivation and participation, and factors influencing these outcomes. Larviciding by LSM committees reduced anopheline larval densities in post-spray sampling compared with pre-spray sampling (P < 0.0001). No differences were observed between anopheline larval densities during pre-spray sampling in LSM villages and those in non-LSM villages (P = 0.282). Knowledge about vector biology and control, and someone's role in LSM motivated community participation in the vector control program. Despite reducing anopheline larval densities in LSM villages, the impact of the community-led LSM could not be detected in our study setting because of low mosquito densities after scale-up of core malaria control interventions. Still, the contributions of the intervention in increasing a community's knowledge of malaria, its risk factors, and its control methods highlight potential benefits of the approach.

Larval source management (LSM) programs for control of malaria vectors are often vertically organized, while there is much potential for involving local communities in program implementation. To address this, we evaluated the entomological impact of community-based application of Bacillus thuringiensis var. israelensis ( Bti ) in a rice irrigation scheme in Ruhuha, Rwanda. A non-randomized trial with control compared a Bti implementation program that was supervised by the project team (ES) with a program that was led and carried out by local rice farming communities (CB). One other area served as a control to assess mosquito populations without Bti application. Entomological surveys were carried out every two weeks and assessed the presence and abundance of the larval, pupal, and adult stages of Anopheles mosquitoes. In ES, the per round reduction in Anopheles larval habitats was estimated at 49%. This reduction was less in CB (28%) and control (22%) although the per round reduction in CB was still significantly higher than in control. Pupal production was almost completely prevented from round 5 (out of 10) onwards in both CB (average habitat occupancy 0.43%) and ES intervention arms (average habitat occupancy 0.27%), whereas pupal occupancy rates were on average 12.8% from round 5 onwards in the control. Emergence of adult mosquitoes from rice fields was thus prevented although this was not directly noticeable in adult An. gambiae populations in houses nearby the rice fields. Together with our earlier work on the willingness to financially contribute to the LSM program and the high perceived safety and acceptance of the Bti product, the current study demonstrates that, in an environment with limited resources, communities could become more engaged in LSM program implementation and contribute directly to malaria vector control in their environment.

For effective sampling of mosquitoes in malaria surveillance programmes, it is essential to include attractive cues in traps. With the aim of implementing a citizen science project on malaria vectors in rural Rwanda, a handmade plastic bottle trap was designed and tested in the field to determine its effectiveness in capturing adult Anopheles gambiae sensu lato, the main malaria vector, and other mosquito species. Carbon dioxide (CO2) and light were used as attractive cues. CO2 was produced by inoculating sugar with yeast and water. Light was emitted from a torch by light-emitting diodes (LEDs). Under field conditions in rural Rwanda, three handmade trap designs were compared to Centers for Disease Control and Prevention miniature light traps (CDC-LT) in houses. The trap baited with yeast produced CO2 and light caught the highest number of mosquitoes compared to the traps baited with light alone or CO2 alone. The number of An. gambiae s.l. in the handmade trap with light and CO2 was approximately 9-10% of the number caught with a CDC light trap. This suggests that about 10 volunteers with a handmade trap could capture a similar-sized sample of An. gambiae as one CDC-LT would collect. Based on these findings, the handmade plastic bottle trap baited with sugar fermenting yeast and light represents an option for inclusion in mosquito surveillance activities in a citizen science context., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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<0.01) and in aquatic habitats with culicine larvae (p<0.01) than in those surrounded by vegetation and not occupied by culicine larvae. Anopheline densities were significantly lower in highly turbid habitats than in clearer habitats (p<0.01). Presence of predators in the aquatic habitats significantly reduced the probability of anopheline larvae being present (p=0.04)., Conclusions: Anopheline larval habitats are widespread in the study area. Presence of bare soil, culicine larvae, predators and the level of turbidity of water are the main determinants of anopheline larval densities in aquatic habitats in Majete, Malawi. While the most productive aquatic habitats should be prioritised, for the most effective control of vectors in the area all available aquatic habitats should be targeted, even those that are not characterized by the identified predictors. Further research is needed to determine whether targeted LSM would be cost-effective when habitat characterisation is included in cost analyses and to establish what methods would make the characterisation of habitats easier., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Background: Application of the larvicide Bacillus thuringiensis var. israelensis (Bti) is a viable complementary strategy for malaria control. Efficacy of Bti is dose-dependent. There is a knowledge gap on the effects of larval exposure to sublethal Bti doses on emerging adult mosquitoes. The present study examined the effect of larval exposure to sublethal doses of Bti on the survival, body size and oviposition rate in adult Anopheles coluzzii., Methods: Third-instar An. coluzzii larvae were exposed to control and sublethal Bti concentrations at LC 20 , LC 50 and LC 70 for 48 h. Surviving larvae were reared to adults under standard colony conditions. Thirty randomly selected females from each treatment were placed in separate cages and allowed to blood feed. Twenty-five gravid females from the blood-feeding cages were randomly selected and transferred into new cages where they were provided with oviposition cups. Numbers of eggs laid in each cage and mortality of all adult mosquitoes were recorded daily. Wing lengths were measured of 570 mosquitoes as a proxy for body size., Results: Exposure to LC 70 Bti doses for 48 h as third-instar larvae reduced longevity of adult An. coluzzii mosquitoes. Time to death was 2.58 times shorter in females exposed to LC 70 Bti when compared to the control females. Estimated mortality hazard rates were also higher in females exposed to the LC 50 and LC 20 treatments, but these differences were not statistically significant. The females exposed to LC 70 concentrations had 12% longer wings than the control group (P < 0.01). No differences in oviposition rate of the gravid females were observed between the treatments., Conclusions: Exposure of An. coluzzii larvae to sublethal Bti doses reduces longevity of resultant adults and is associated with larger adult size and unclear effect on oviposition. These findings suggest that anopheline larval exposure to sublethal Bti doses, though not recommended, could reduce vectorial capacity for malaria vector populations by increasing mortality of resultant adults.

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.

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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Background: Insecticide resistance is greatly hampering current efforts to control malaria and therefore alternative methods are needed. Entomopathogenic fungi have been proposed as an alternative with a special focus on the cosmopolitan species Beauveria bassiana. However, few studies have analysed the effects of natural variation within fungal isolates on mosquito survival, and the implications and possible exploitation for malaria control., Methods: Laboratory bioassays were performed on adult female mosquitoes (Anopheles coluzzii) with spores from 29 isolates of B. bassiana, originating from different parts of the world. In addition, phenotypic characteristics of the fungal isolates such as sporulation, spore size and growth rate were studied to explore their relationship with virulence., Results: All tested isolates of B. bassiana killed An. coluzzii mosquitoes, and the rate at which this happened differed significantly among the isolates. The risk of mosquitoes dying was around ten times higher when they were exposed to the most virulent as compared to the least virulent isolate. There was significant variation among isolates in spore size, growth rate and sporulation, but none of these morphological characteristics were correlated, and thus predictive, for the ability of the fungal isolate to kill malaria mosquitoes., Conclusions: This study shows that there is a wide natural variation in virulence of isolates of B. bassiana, and that selecting an appropriate fungal isolate is highly relevant in killing and thus controlling malaria mosquitoes, particularly if used as part of an integrated vector management strategy. Also, the wide variation observed in virulence offers the opportunity to better understand the molecular and genetic mechanisms that drive this variation and thus to address the potential development of resistance against entomopathogenic fungi.

Entomopathogenic fungi have shown to be effective in biological control of both larval and adult stages of malaria mosquitoes. However, a small fraction of mosquitoes is still able to emerge after treatment with fungus during the larval stage. It remains unclear whether fitness of these adults is affected by the treatment during the larval stage and whether they are still susceptible for another treatment during the adult stage. Therefore, we tested the effects of larval exposure to the entomopathogenic fungus Beauveria bassiana on fitness of surviving Anopheles stephensi females. Furthermore, we tested whether larval exposed females were still susceptible to re-exposure to the fungus during the adult stage. Sex ratio, survival and reproductive success were compared between non-exposed and larval exposed A. stephensi. Comparisons were also made between survival of non-exposed and larval exposed females that were re-exposed to B. bassiana during the adult stage. Larval treatment did not affect sex ratio of emerging mosquitoes. Larval exposed females that were infected died significantly faster and laid equal numbers of eggs from which equal numbers of larvae hatched, compared to non-exposed females. Larval exposed females that were uninfected had equal survival, but laid a significantly larger number of eggs from which a significantly higher number of larvae hatched, compared to non-exposed females. Larval exposed females which were re-exposed to B. bassiana during the adult stage had equal survival as females exposed only during the adult stage. Our results suggest that individual consequences for fitness of larval exposed females depended on whether a fungal infection was acquired during the larval stage. Larval exposed females remained susceptible to re-exposure with B. bassiana during the adult stage, indicating that larval and adult control of malaria mosquitoes with EF are compatible., (Copyright © 2014. Published by Elsevier Inc.)

Flight behavior of insecticide-resistant and susceptible malaria mosquitoes approaching deltamethrin-treated nets was examined using a wind tunnel. Behavior was linked to resulting health status (dead or alive) using comparisons between outcomes from free-flight assays and standard World Health Organization (WHO) bioassays. There was no difference in response time, latency time to reach the net, or spatial distribution in the wind tunnel between treatments. Unaffected resistant mosquitoes spent less time close to (< 30 cm) treated nets. Nettings that caused high knockdown or mortality in standard WHO assays evoked significantly less mortality in the wind tunnel; there was no excitorepellent effect in mosquitoes making contact with the nettings in free flight. This study shows a new approach to understanding mosquito behavior near insecticidal nets. The methodology links free-flight behavior to mosquito health status on exposure to nets. The results suggest that behavioral assays can provide important insights for evaluation of insecticidal effects on disease vectors., (© The American Society of Tropical Medicine and Hygiene.)

Background: Control of mosquitoes that transmit malaria has been the mainstay in the fight against the disease, but alternative methods are required in view of emerging insecticide resistance. Entomopathogenic fungi are candidate alternatives, but to date, few trials have translated the use of these agents to field-based evaluations of their actual impact on mosquito survival and malaria risk. Mineral oil-formulations of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were applied using five different techniques that each exploited the behaviour of malaria mosquitoes when entering, host-seeking or resting in experimental huts in a malaria endemic area of rural Tanzania., Results: Survival of mosquitoes was reduced by 39-57% relative to controls after forcing upward house-entry of mosquitoes through fungus treated baffles attached to the eaves or after application of fungus-treated surfaces around an occupied bed net (bed net strip design). Moreover, 68 to 76% of the treatment mosquitoes showed fungal growth and thus had sufficient contact with fungus treated surfaces. A population dynamic model of malaria-mosquito interactions shows that these infection rates reduce malaria transmission by 75-80% due to the effect of fungal infection on adult mortality alone. The model also demonstrated that even if a high proportion of the mosquitoes exhibits outdoor biting behaviour, malaria transmission was still significantly reduced., Conclusions: Entomopathogenic fungi strongly affect mosquito survival and have a high predicted impact on malaria transmission. These entomopathogens represent a viable alternative for malaria control, especially if they are used as part of an integrated vector management strategy.

Background: Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions., Methods: This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions., Results: The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5 × for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%., Conclusions: This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate.

Physiological characteristics of insects can influence their susceptibility to fungal infection of which age and nutritional status are among the most important. An understanding of host-pathogen interaction with respect to these physiological characteristics of the host is essential if we are to develop fungal formulations capable of reducing malaria transmission under field conditions. Here, two independent bioassays were conducted to study the effect of age and blood-feeding status on fungal infection and survival of Anopheles gambiae s.s. Giles. Mosquitoes were exposed to 2 × 10(10) conidia m(-2) of oil-formulated Metarhizium anisopliae ICIPE-30 and of Beauveria bassiana I93-825, respectively, and their survival was monitored daily. Three age groups of mosquitoes were exposed, 2-4, 5-8, and 9-12 days since emergence. Five groups of different feeding status were exposed: non-blood-fed, 3, 12, 36, and 72 h post-blood feeding. Fungal infection reduced the survival of mosquitoes regardless of their age and blood-feeding status. Although older mosquitoes died relatively earlier than younger ones, age did not tend to affect mosquito susceptibility to fungal infection. Non-blood-fed mosquitoes were more susceptible to fungus infection compared to all categories of blood-fed mosquitoes, except for those exposed to B. bassiana 72 h post-blood feeding. In conclusion, formulations of M. anisopliae and B. bassiana can equally affect mosquitoes of different age classes, with them being relatively more susceptible to fungus infection when non-blood-fed.

Background: Entomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of Anopheles gambiae sensu stricto, and to test a potential field application method with respect to the viability and virulence of two fungal species, Methods: Pieces of white polyester netting were dipped in Metarhizium anisopliae ICIPE-30 or Beauveria bassiana IMI391510 mineral oil suspensions. These were kept at 27 +/- 1 degrees C, 80 +/- 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of An. gambiae s.s., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net., Results: The resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, B. bassiana was significantly more virulent than M. anisopliae for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension., Conclusions: The insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.

Background: Entomopathogenic fungi have shown great potential for the control of adult malaria vectors. However, their ability to control aquatic stages of anopheline vectors remains largely unexplored. Therefore, how larval characteristics (Anopheles species, age and larval density), fungus (species and concentration) and environmental effects (exposure duration and food availability) influence larval mortality caused by fungus, was studied., Methods: Laboratory bioassays were performed on the larval stages of Anopheles gambiae and Anopheles stephensi with spores of two fungus species, Metarhizium anisopliae and Beauveria bassiana. For various larval and fungal characteristics and environmental effects the time to death was determined and survival curves established. These curves were compared by Kaplan Meier and Cox regression analyses., Results: Beauveria bassiana and Metarhizium anisopliae caused high mortality of An. gambiae and An. stephensi larvae. However, Beauveria bassiana was less effective (Hazard ratio (HR) <1) compared to Metarhizium anisopliae. Anopheles stephensi and An. gambiae were equally susceptible to each fungus. Older larvae were less likely to die than young larvae (HR < 1). The effect of increase in fungus concentration on larval mortality was influenced by spore clumping. One day exposure to fungal spores was found to be equally effective as seven days exposure. In different exposure time treatments 0 - 4.9% of the total larvae, exposed to fungus, showed infection at either the pupal or adult stage. Mortality rate increased with increasing larval density and amount of available food., Conclusions: This study shows that both fungus species have potential to kill mosquitoes in the larval stage, and that mortality rate depends on fungus species itself, larval stage targeted, larval density and amount of nutrients available to the larvae. Increasing the concentration of fungal spores or reducing the exposure time to spores did not show a proportional increase and decrease in mortality rate, respectively, because the spores clumped together. As a result spores did not provide uniform coverage over space and time. It is, therefore, necessary to develop a formulation that allows the spores to spread over the water surface. Apart from formulation appropriate delivery methods are also necessary to avoid exposing non-target organisms to fungus.

Background: Although the effects of rainfall on the population dynamics of the malaria vector Anopheles gambiae have been studied in great detail, the effects of dry periods on its survival remain less clear., Methods: The effects of drying conditions were simulated by creating desiccated habitats, which consisted of trays filled with damp soil. Experiments were performed in these trays to (i) test the ability of An. gambiae sensu stricto eggs to hatch on damp soil and for larvae to reach an artificial breeding site at different distances of the site of hatching and (ii) to record survival of the four larval stages of An. gambiae s.s. when placed on damp soil., Results: Eggs of An. gambiae s.s. hatched on damp soil and emerging larvae were capable of covering a distance of up to 10 cm to reach surface water enabling further development. However, proportions of larvae reaching the site decreased rapidly with increasing distance. First, second and third-instar larvae survived on damp soil for an estimated period of 64, 65 and 69 hrs, respectively. Fourth-instar larvae survived significantly longer and we estimated that the maximum survival time was 113 hrs., Conclusion: Short-term survival of aquatic stages of An. gambiae on wet soil may be important and adaptive when considering the transient nature of breeding sites of this species in sub-Saharan Africa. In addition, the results suggest that, for larval vector control methods to be effective, habitats should remain drained for at least 5 days to kill all larvae (e.g. in rice fields) and habitats that recently dried up should be treated as well, if larvicidal agents are applied.

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

The two main interventions presently being deployed for control of malaria vectors, that is, long-lasting insecticide-treated bed nets (LLINs) and indoor residual spraying (IRS) involve the use of chemical insecticides and target adult mosquitoes. Meanwhile, the potential of larval control is increasingly being acknowledged for the reduction of insecticide-resistant and/or exophagicAnophelespopulations. Larval control has proven to be cost-effective and ideal for localities where mosquito-breeding sites are well defined and approachable. Utilising biological control tools to control anopheline larvae can lower the problem of resistance development, a common feature of chemical control. Fortunately, there are many options of biological larval control tools. Besides their direct impact on mortality, the effects of these tools can reach beyond the larval stage. Anopheline adults that develop from larvae exposed to biological control tools, such as entomopathogenic fungi, show reduced longevity, fecundity and susceptibility toPlasmodiuminfections. Combining two or more larval control tools can increase their efficacy against anopheline larvae. However, despite the identification of larval control potential in the laboratory, and in few cases in the field, many potential biological control tools have not been utilised to their fullest extent. This review provides an overview of the existing and potential biological larval control options for malaria vectors and discusses the advantages and requirements to develop them for malaria vector control. [ABSTRACT FROM AUTHOR]