Considerations for first field trials of low-threshold gene drive for malaria vector control.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a 'causal pathway' would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

Challenges of proper disposal of old long-lasting insecticidal nets and its alternative uses in rural south-eastern Tanzania.

INTRODUCTION: Insecticide-treated nets (ITNs), specifically long-lasting insecticidal nets (LLINs), are the most commonly used, scalable, and cost-effective tools for controlling malaria transmission in sub-Saharan Africa. However, the multiple alternative uses of retired LLINs have been associated with poor disposal practices. The World Health Organization (WHO) has provided guidelines and recommendations for the proper management of worn-out LLINs. This study assessed the existing alternative uses and disposal practices of old LLINs. METHODS: An explanatory sequential mixed-methods approach was used to assess LLINs existing alternative uses, disposal practices, knowledge, and perceptions regarding WHO recommendations on proper disposal of old LLINs among stakeholders in Kilombero and Ulanga districts, south-eastern Tanzania. A survey questionnaire was administered to 384 participants. Furthermore, the study employed focus group discussions (FGD) and key informant interviews (KII) to elucidate responses regarding existing disposal practices, associated challenges, and alternative uses of LLINs. The insights derived from both study components were subsequently used for inferential analysis. RESULTS: The major challenge influencing the proper disposal of LLINs was limited awareness of how to properly dispose of them. Of the 384 people surveyed, 97.0% were not aware of the WHO recommendations for the proper disposal of old LLINs. All key informants were unaware of the WHO guidelines for proper disposal of old LLINs. The common methods used to dispose of LLINs were burning (30.7%), disposing them into garbage pits (14.8%), and alternative uses (12.2%). Of the 239 respondents with LLINs, 41.0% had alternative use, while 59.0% had no alternative use. The common alternative uses were ropes for tying or covering items (20.9%), garden fencing (7.5%), chicken coops (5.0%), and 7.5% for other minor alternative uses. CONCLUSION: Strengthening awareness and education on proper LLIN disposal practices among community members and key stakeholders is essential for enhancing malaria control efforts and preventing environmental pollution.

Community perception of the autodissemination of pyriproxyfen for controlling malaria vectors in south-eastern Tanzania.

BACKGROUND: The efficacy of the autodissemination of pyriproxyfen to control malaria vectors has been demonstrated under semi field environment in Tanzania. However, the information on how best communities should be engaged for its routine and large-scale adoption are lacking. This study assessed the community's level of knowledge, perceptions, acceptability of the autodissemination of pyriproxyfen, and the perceived risks on the safety of pyriproxyfen on the environment. METHODS: This was a concurrent mixed methods study, comprised of a community-based survey of 400 household representatives and eight focus group discussions (FGDs). The study was conducted in two villages in Mlimba district in south-eastern Tanzania between June and August 2022. For the quantitative data analysis, descriptive statistics were applied using R software, while inductive approach was used for qualitative data analysis, using NVivo software. RESULTS: Knowledge on autodissemination of pyriproxyfen approach was found to be relatively low among both the FGD respondents and surveyed community members (36%, n = 144). Nevertheless, when it was explained to them, the envisioned community support for the autodissemination approach was relatively high (97%, n = 388). One of the major perceived benefits of the autodissemination of pyriproxyfen was the reduction of malaria-transmitting mosquitoes and associated malaria transmission. Environmental impact of pyriproxyfen on non-target organisms and health risk to children were among the major concerns. When provided with information on the safety and its utilization particularly through autodissemination approach, 93.5% (n = 374) of the survey respondents said that they would allow the PPF-contaminated pots to be placed around their homes. Similarly, FGD respondents were receptive towards the autodissemination of pyriproxyfen, but emphasized on the need for raising awareness among community members before related field trials. CONCLUSION: This study indicates a low knowledge but high support for scaling up of the autodissemination of pyriproxyfen as a complementary tool for malaria control in rural Tanzania. The Findings of this study suggest that community sensitization activities are required to improve the community's acceptability and trust of the approach before respective field trials.

Sterilized Anopheles funestus can autodisseminate sufficient pyriproxyfen to the breeding habitat under semi-field settings.

BACKGROUND: Anopheles funestus, the main malaria vector, prefer to oviposit in permanent and/or semi-permanent breeding habitats located far from human dwellings. Difficulties in identifying and accessing these habitats jeopardize the feasibility of conventional larviciding. In this way, a semi-field study was conducted to assess the potential of autodissemination of pyriproxyfen (PPF) by An. funestus for its control. METHODS: The study was conducted inside a semi-field system (SFS). Therein, two identical separate chambers, the treatment chamber with a PPF-treated clay pot (0.25 g AI), and the control chamber with an untreated clay pot. In both chambers, one artificial breeding habitat made of a plastic basin with one litre of water was provided. Three hundred blood-fed female An. funestus aged 5-9 days were held inside untreated and treated clay pots for 30 min and 48 h before being released for oviposition. The impact of PPF on adult emergence, fecundity, and fertility through autodissemination and sterilization effects were assessed by comparing the treatment with its appropriate control group. RESULTS: Mean (95% CI) percentage of adult emergence was 15.5% (14.9-16.1%) and 70.3% (69-71%) in the PPF and control chamber for females exposed for 30 min (p < 0.001); and 19% (12-28%) and 95% (88-98%) in the PPF and control chamber for females exposed for 48 h (p < 0.001) respectively. Eggs laid by exposed mosquitoes and their hatch rate were significantly reduced compared to unexposed mosquitoes (p < 0.001). Approximately, 90% of females exposed for 48 h retained abnormal ovarian follicles and only 42% in females exposed for 30 min. CONCLUSION: The study demonstrated sterilization and adult emergence inhibition via autodissemination of PPF by An. funestus. Also, it offers proof that sterilized An. funestus can transfer PPF to prevent adult emergence at breeding habitats. These findings warrant further assessment of the autodissemination of PPF in controlling wild population of An. funestus, and highlights its potential for complementing long-lasting insecticidal nets.

Current and future opportunities of autodissemination of pyriproxyfen approach for malaria vector control in urban and rural Africa.

Despite the progress made in reducing malaria burden, new ways to address the increasing challenges of insecticide resistance and the invasion and spread of exotic malaria vectors such as Anopheles stephensi in Africa are urgently needed. While African countries are adopting larviciding as a complementary intervention for malaria vector control, the autodissemination technology has the potential to overcome barriers associated with the identification and treatment of prolific habitats that impede conventional larviciding approaches in rural settings. The autodissemination technology as a "lure and release" strategy works by exploiting the resting behavior of gravid mosquitoes to transfer lethal concentration of biological or chemical insecticide such as pyriproxyfen (PPF), an insect growth regulator (IGRs) to their oviposition sites and result in adult emergence inhibition. Despite the evidence of the autodissemination approach to control other mosquito-borne diseases, there is growing and promising evidence for its use in controlling malaria vectors in Africa, which highlights the momentous research that needs to be sustained. This article reviews the evidence for efficacy of the autodissemination approach using PPF and discusses its potential as efficient and affordable complementary malaria vector control intervention in Africa. In the previous studies that were done in controlled semi-field environments, autodissemination with PPF demonstrated its potential in reducing densities of captive population of malaria vectors such as Anopheles gambiae and Anopheles arabiensis. Of importance, empirical evidence and biology-informed mathematical models to demonstrate the utility of the autodissemination approach to control wild populations of malaria vectors under field environment either alone or in combination with other tools are underway. Among others, the key determining factors for future introduction of this approach at scale is having scalable autodissemination devices, optimized PPF formulations, assess its integration/complementarity to existing conventional larviciding, and community perception and acceptance of the autodissemination approach.

Susceptibility status of major malaria vectors to novaluron, an insect growth regulator South-Eastern Tanzania.

Introduction: application of Insect Growth Regulator (IGR) such as pyriproxyfen has shown a promising result in controlling malaria transmitting mosquitoes through autodissemination technique. Novaluron that inhibits the chitin development at mosquito larval stage present a promising candidate IGR for rotation with pyriproxyfen to prevent a chance of resistance development. This study assessed the susceptibility of immature stages of Anopheles arabiensis, Anopheles gambiae and Anopheles funestus to novaluron. Methods: susceptibility bioassays using technical grade novaluron (98% active ingredient) were performed inside the semi-field system using first instar larvae of Anopheles species. For each tested species, a total of 1500 larvae were used in the bioassay. Concentration range of 0.01 mg/l to 2 mg/l of novaluron were tested to establish Lethal Concentration (LC) sufficient to kills 50%, 90% and 99% of the exposed larvae by using log-dose response analysis. Results: of the tested mosquitoes, Anopheles gambiae were highly susceptible to novaluron followed by An. Arabiensis and then An. funestus. Lethal concentrations, LC50, LC90 and LC99 (95%CI) in mg/l for An. gambiae were 0.018, 0.332 and 2.001 respectively. For An. arabiensis were 0.026, 0.546 and 2.013; and for An. funestus were 0.032, 1.00 and 5.580. High larval mortality was recorded at high concentration (2mg/L), with 80% mortality within 3 days post exposure. Conclusion: the study demonstrates the efficacy of novaluron in controlling Anopheles mosquito species at immature stages via larval mortality. These findings warrant further testing of novaluron for autodissemination by different vector species for its inclusion in rotation to prevent development of resistance.

The ability of Anopheles funestus and A. arabiensis to penetrate LLINs and its effect on their mortality.

Background: Variation in mosquito body size and the ability to penetrate long-lasting insecticide-treated nets (LLINs) remains unknown. This study evaluated the ability of Anopheles funestus and A. arabiensis to penetrate commercially available treated and untreated bednets and how this behaviour affects mosquito mortality. Methods: Three types of LLINs; DawaPlus 2.0, PermaNet 2.0, Olyset 2.0, and untreated (Safi Net) were tested inside a semi-field system. One hundred 3-5-day-old and non-starved female A. funestus and A. arabiensis were released in a chamber with a sleeping adult volunteer under a treated or untreated bednet. Mosquitoes that penetrated inside the nets were collected every two hours using a mouth aspirator. Live mosquitoes were put in paper cups, fed on glucose ad libitum and their mortality rate was monitored for 48 h. Results: The ability of A. funestus to penetrate treated and untreated bednets was significantly higher than for A. arabiensis for all three LLIN net types ( P<0.001). For both species the penetration rate was higher for untreated bednets than treated ones except for the Olyset net. Regardless of the assessed mosquito species, all the mosquitoes that penetrated the net, successfully blood-fed on the sleeping volunteer. Compared to A. arabiensis, significant mortality was recorded for A. funestus that were caught inside Olyset nets within 48 hrs of monitoring ( P<0.001). Conclusions: These findings demonstrate the ability of A. funestus and A. arabiensis mosquitoes to penetrate the human-occupied treated and untreated bednets. Despite this ability, mosquitoes that penetrated the bednet succumbed to death within two days.

Infrastructure alone cannot ensure resilience to weather events in drinking water supplies.

Climate resilient water supplies are those that provide access to drinking water that is sustained through seasons and through extreme events, and where good water quality is also sustained. While surface and groundwater quality are widely understood to vary with rainfall, there is a gap in the evidence on the impact of weather and extremes in rainfall and temperature on drinking water quality, and the role of changes in water system management. A three-country (Bangladesh, Nepal and Tanzania) observational field study tracked 2353 households clustered around 685 water sources across seven different geographies over 14 months. Water quality (E. coli) data was modelled using GEE to account for clustering effects and repeated measures at households. All types of infrastructure were vulnerable to changes in weather, with differences varying between geographies; protected boreholes provided the greatest protection at the point of collection (PoC). Water quality at the point of use (PoU) was vulnerable to changes in weather, through changes in PoC water quality as well as changes in management behaviours, such as safe storage, treatment and cleaning. This is the first study to demonstrate the impact of rainfall and temperature extremes on water quality at the PoC, and the role that weather has on PoU water quality via management behaviours. Climate resilience for water supplies needs to consider the infrastructure as well as the management decisions that are taking place at a community and household level.

Proceedings of an expert workshop on community agreement for gene drive research in Africa - Co-organised by KEMRI, PAMCA and Target Malaria.

Gene drive research is progressing towards future field evaluation of modified mosquitoes for malaria control in sub-Saharan Africa. While many literature sources and guidance point to the inadequacy of individual informed consent for any genetically modified mosquito release, including gene drive ones, (outside of epidemiological studies that might require blood samples) and at the need for a community-level decision, researchers often find themselves with no specific guidance on how that decision should be made, expressed and by whom. Target Malaria, the Kenya Medical Research Institute and the Pan African Mosquito Control Association co-organised a workshop with researchers and practitioners on this topic to question the model proposed by Target Malaria in its research so far that involved the release of genetically modified sterile male mosquitoes and how this could be adapted to future studies involving gene drive mosquito releases for them to offer reflections about potential best practices. This paper shares the outcomes of that workshop and highlights the remaining topics for discussion before a comprehensive model can be designed.

Using pastoralist community knowledge to locate and treat dry-season mosquito breeding habitats with pyriproxyfen to control Anopheles gambiae s.l. and Anopheles funestus s.l. in rural Tanzania.

Fundamentally, larviciding with pyriproxyfen (PPF) has potential to complement Long Lasting Insecticide Nets (LLINs) and indoor residual sprays (IRS) in settings where resistance to pyrethroids and residual malaria transmission exist. In this study, we evaluated the field effectiveness of larviciding using PPF to reduce dry season productivity of mosquito breeding habitats that were located by pastoralists within the study area. Using pastoralist knowledge, dry season breeding habitats in Mofu village rural Tanzania were located and monitored for larval productivity for a period of 8 months before PPF intervention. During the intervention, six out of twelve breeding habitats were treated with Sumilarv 0.5G PPF granules. The impact of deposited PPF was monitored by recording emergence inhibition of larvae collected from treated habitats compared to the appropriate control group for a period of three months and half post-intervention. During baseline, the average proportion (+SD) of adult emerged was similar between two clusters, with (0.89 + 0.22) for the control cluster and (0.93 + 0.16) for the treatment cluster of breeding habitats. Following treatment with PPF, the average proportion (+SD) of adult emerged in the treated breeding habitats was significantly low (0.096 + 0.22) compared to adults that emerged from larvae in the untreated habitats (0.99 + 0.22) (p < 0.0001). Of all emerged adults, approximately 94% were An. gambiae s.l. and the remaining 6% were An. funestus s.l. This is the first study demonstrating the usefulness of engaging pastoralist community to locate and identify hard to find mosquito breeding habitats. Reduced productivity of the targeted habitats with PPF offers prospect of implementing PPF larviciding in dry season when habitats are few and permanent to control mosquito population in rural settings.

A retrospective study on the epidemiology and trends of road traffic accidents, fatalities and injuries in three Municipalities of Dar es Salaam Region, Tanzania between 2014-2018.

INTRODUCTION: Over 90% of injuries and deaths still occur in low and middle-income countries like Tanzania due to Road traffic accidents. Available literature indicates that Tanzania suffers massive human and economic losses every year from RTAs despite several interventions that have been made to curb this scourge. To gain an insight into the current state of RTAs we examined the pre- historical case fatality rates from RTAs in Ilala and two other municipalities (Kinondoni and Temeke) in Dar es Salaam Region, Tanzania. METHODS: We conducted a retrospective study using the secondary data on road accidents from Road Accident Information System (RAIS) for the period 2014 to 2018. RESULTS: A total of 6,772 road traffic injuries were reported between 2014 and 2018 and the study recorded the highest RTAs in the year 2014 as compared to the other years within the study period. The death rate from RTAs in Ilala Municipality alone was 36.4 per 100,000 population. About 28% of the total fatalities were recorded among the pedestrians, and there was a significant difference (P < 0.05) in the RTAs among the other road users. CONCLUSION: The study recommends the improvement of road transport infrastructure to ensure safety for all the road users by implementing the existing policies, strengthening the enforcement of existing legislation and introducing express penalties on a real-time basis. We encourage the use of this data to develop strategies in Tanzania that protect pedestrians and other vulnerable road users from RTAs.

Aquatic habitats of the malaria vector Anopheles funestus in rural south-eastern Tanzania.

BACKGROUND: In rural south-eastern Tanzania, Anopheles funestus is a major malaria vector, and has been implicated in nearly 90% of all infective bites. Unfortunately, little is known about the natural ecological requirements and survival strategies of this mosquito species. METHODS: Potential mosquito aquatic habitats were systematically searched along 1000 m transects from the centres of six villages in south-eastern Tanzania. All water bodies were geo-referenced, characterized and examined for presence of Anopheles larvae using standard 350 mLs dippers or 10 L buckets. Larvae were collected for rearing, and the emergent adults identified to confirm habitats containing An. funestus. RESULTS: One hundred and eleven habitats were identified and assessed from the first five villages (all < 300 m altitude). Of these, 36 (32.4%) had An. funestus co-occurring with other mosquito species. Another 47 (42.3%) had other Anopheles species and/or culicines, but not An. funestus, and 28 (25.2%) had no mosquitoes. There were three main habitat types occupied by An. funestus, namely: (a) small spring-fed pools with well-defined perimeters (36.1%), (b) medium-sized natural ponds retaining water most of the year (16.7%), and (c) slow-moving waters along river tributaries (47.2%). The habitats generally had clear waters with emergent surface vegetation, depths > 0.5 m and distances < 100 m from human dwellings. They were permanent or semi-permanent, retaining water most of the year. Water temperatures ranged from 25.2 to 28.8 °C, pH from 6.5 to 6.7, turbidity from 26.6 to 54.8 NTU and total dissolved solids from 60.5 to 80.3 mg/L. In the sixth village (altitude > 400 m), very high densities of An. funestus were found along rivers with slow-moving clear waters and emergent vegetation. CONCLUSION: This study has documented the diversity and key characteristics of aquatic habitats of An. funestus across villages in south-eastern Tanzania, and will form an important basis for further studies to improve malaria control. The observations suggest that An. funestus habitats in the area can indeed be described as fixed, few and findable based on their unique characteristics. Future studies should investigate the potential of targeting these habitats with larviciding or larval source management to complement malaria control efforts in areas dominated by this vector species.

Autodissemination of pyriproxyfen suppresses stable populations of Anopheles arabiensis under semi-controlled settings.

BACKGROUND: Autodissemination of pyriproxyfen (PPF), i.e. co-opting adult female mosquitoes to transfer the insect growth regulator, pyriproxyfen (PPF) to their aquatic habitats has been demonstrated for Aedes and Anopheles mosquitoes. This approach, could potentially enable high coverage of aquatic mosquito habitats, including those hard to locate or reach via conventional larviciding. This study demonstrated impacts of autodissemination in crashing a stable and self-sustaining population of the malaria vector, Anopheles arabiensis under semi-field conditions in Tanzania. METHODS: Self-propagating populations of An. arabiensis were established inside large semi-field cages. Larvae fed on naturally occurring food in 20 aquatic habitats in two study chambers (9.6 × 9.6 m each), while emerging adults fed on tethered cattle. The mosquito population was monitored using emergence traps and human landing catches, each time returning captured adults into the chambers. Once the population was stable (after 23 filial generations), PPF dissemination devices (i.e. four clay pots each treated with 0.2-0.3 g PPF) were introduced into one of the chambers (treatment) and their impact monitored in parallel with untreated chamber (control). RESULTS: Daily adult emergence was similar between control and treatment chambers, with average (± SE) of 14.22 ± 0.70 and 12.62 ± 0.74 mosquitoes/trap, respectively, before treatment. Three months post-treatment, mean number of adult An. arabiensis emerging from the habitats was 5.22 ± 0.42 in control and 0.14 ± 0.04 in treatment chambers. This was equivalent to > 97% suppression in treatment chamber without re-treatment of the clay pots. Similarly, the number of mosquitoes attempting to bite volunteers inside the treatment chamber decreased to zero, 6 months post-exposure (i.e. 100% suppression). In contrast, biting rates in control rose to 53.75 ± 3.07 per volunteer over the same period. CONCLUSION: These findings demonstrate effective suppression of stable populations of malaria vectors using a small number of simple autodissemination devices, from which adult mosquitoes propagated pyriproxyfen to contaminate aquatic habitats in the system. This is the first proof that autodissemination can amplify treatment coverage and deplete malaria vector populations. Field trials are necessary to validate these results, and assess impact of autodissemination as a complementary malaria intervention.

Understanding the Impacts of Short-Term Climate Variability on Drinking Water Source Quality: Observations From Three Distinct Climatic Regions in Tanzania.

Climate change is expected to increase waterborne diseases especially in developing countries. However, we lack understanding of how different types of water sources (both improved and unimproved) are affected by climate change, and thus, where to prioritize future investments and improvements to maximize health outcomes. This is due to limited knowledge of the relationships between source water quality and the observed variability in climate conditions. To address this gap, a 20-month observational study was conducted in Tanzania, aiming to understand how water quality changes at various types of sources due to short-term climate variability. Nine rounds of microbiological water quality sampling were conducted for Escherichia coli and total coliforms, at three study sites within different climatic regions. Each round included approximately 233 samples from water sources and 632 samples from households. To identify relationships between water quality and short-term climate variability, Bayesian hierarchical modeling was adopted, allowing these relationships to vary with source types and sampling regions to account for potentially different physical processes. Across water sources, increases in E. coli/total coliform levels were most closely related to increases in recent heavy rainfall. Our key recommendations to future longitudinal studies are (a) demonstrated value of high sampling frequency and temporal coverage (a minimum of 3 years) especially during wet seasons; (b) utility of the Bayesian hierarchical models to pool data from multiple sites while allowing for variations across space and water sources; and (c) importance of a multidisciplinary team approach with consistent commitment and sharing of knowledge.

Using Stable Isotopes of Carbon and Nitrogen to Mark Wild Populations of Anopheles and Aedes Mosquitoes in South-Eastern Tanzania.

BACKGROUND: Marking wild mosquitoes is important for understanding their ecology, behaviours and role in disease transmission. Traditional insect marking techniques include using fluorescent dyes, protein labels, radioactive labels and tags, but such techniques have various limitations; notably low marker retention and inability to mark wild mosquitoes at source. Stable isotopes are gaining wide spread use for non-invasive marking of arthropods, permitting greater understanding of mosquito dispersal and responses to interventions. We describe here a simple technique for marking naturally-breeding malaria and dengue vectors using stable isotopes of nitrogen (15N) and carbon (13C), and describe potential field applications. METHODS: We created man-made aquatic mosquito habitats and added either 15N-labelled potassium nitrate or 13C-labelled glucose, leaving non-adulterated habitats as controls. We then allowed wild mosquitoes to lay eggs in these habitats and monitored their development in situ. Pupae were collected promptly as they appeared and kept in netting cages. Emergent adults (in pools of ~4 mosquitoes/pool) and individually stored pupae were desiccated and analysed using Isotope Ratio Mass Spectrometry (IRMS). FINDINGS: Anopheles gambiae s.l and Aedes spp. from enriched 13C and enriched 15N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ15N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ15N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ13C for enriched females and males were 36.08±5.28 and 38.5±6.86, compared to -4.3±0.2 and -7.9±3.6 in controls respectively. Mean δ15N and δ13C was significantly higher in any pool containing at least one enriched mosquito compared to pools with all unenriched mosquitoes, P<0.001. In all cases, there were variations in standardized isotopic ratios between mosquito species. CONCLUSION: Enrichment of semi-natural mosquito larval habitats with stable isotopes of nitrogen and carbon resulted in effective marking of Anopheles and Aedes mosquitoes colonizing these habitats. This approach can significantly enhance studies on mosquito eco-physiology, dispersal, pathogen transmission and responses to control measures.

Most outdoor malaria transmission by behaviourally-resistant Anopheles arabiensis is mediated by mosquitoes that have previously been inside houses.

BACKGROUND: Anopheles arabiensis is stereotypical of diverse vectors that mediate residual malaria transmission globally, because it can feed outdoors upon humans or cattle, or enter but then rapidly exit houses without fatal exposure to insecticidal nets or sprays. METHODS: Life histories of a well-characterized An. arabiensis population were simulated with a simple but process-explicit deterministic model and relevance to other vectors examined through sensitivity analysis. RESULTS: Where most humans use bed nets, two thirds of An. arabiensis blood feeds and half of malaria transmission events were estimated to occur outdoors. However, it was also estimated that most successful feeds and almost all (>98 %) transmission events are preceded by unsuccessful attempts to attack humans indoors. The estimated proportion of vector blood meals ultimately obtained from humans indoors is dramatically attenuated by availability of alternative hosts, or partial ability to attack humans outdoors. However, the estimated proportion of mosquitoes old enough to transmit malaria, and which have previously entered a house at least once, is far less sensitive to both variables. For vectors with similarly modest preference for cattle over humans and similar ability to evade fatal indoor insecticide exposure once indoors, >80 % of predicted feeding events by mosquitoes old enough to transmit malaria are preceded by at least one house entry event, so long as ≥40 % of attempts to attack humans occur indoors and humans outnumber cattle ≥4-fold. CONCLUSIONS: While the exact numerical results predicted by such a simple deterministic model should be considered only approximate and illustrative, the derived conclusions are remarkably insensitive to substantive deviations from the input parameter values measured for this particular An. arabiensis population. This life-history analysis, therefore, identifies a clear, broadly-important opportunity for more effective suppression of residual malaria transmission by An. arabiensis in Africa and other important vectors of residual transmission across the tropics. Improved control of predominantly outdoor residual transmission by An. arabiensis, and other modestly zoophagic vectors like Anopheles darlingi, which frequently enter but then rapidly exit from houses, may be readily achieved by improving existing technology for killing mosquitoes indoors.

A generic schema and data collection forms applicable to diverse entomological studies of mosquitoes.

BACKGROUND: Standardized schemas, databases, and public data repositories are needed for the studies of malaria vectors that encompass a remarkably diverse array of designs and rapidly generate large data volumes, often in resource-limited tropical settings lacking specialized software or informatics support. RESULTS: Data from the majority of mosquito studies conformed to a generic schema, with data collection forms recording the experimental design, sorting of collections, details of sample pooling or subdivision, and additional observations. Generically applicable forms with standardized attribute definitions enabled rigorous, consistent data and sample management with generic software and minimal expertise. Forms use now includes 20 experiments, 8 projects, and 15 users at 3 research and control institutes in 3 African countries, resulting in 11 peer-reviewed publications. CONCLUSION: We have designed generic data schema that can be used to develop paper or electronic based data collection forms depending on the availability of resources. We have developed paper-based data collection forms that can be used to collect data from majority of entomological studies across multiple study areas using standardized data formats. Data recorded on these forms with standardized formats can be entered and linked with any relational database software. These informatics tools are recommended because they ensure that medical entomologists save time, improve data quality, and data collected and shared across multiple studies is in standardized formats hence increasing research outputs.

Effects of a new outdoor mosquito control device, the mosquito landing box, on densities and survival of the malaria vector, Anopheles arabiensis, inside controlled semi-field settings.

BACKGROUND: The significance of malaria transmission occurring outdoors has risen even in areas where indoor interventions such as long-lasting insecticidal nets and indoor residual spraying are common. The actual contamination rates and effectiveness of recently developed outdoor mosquito control device, the mosquito landing box (MLB), on densities and daily survival of host-seeking laboratory Anopheles arabiensis, which readily bites humans outdoors was demonstrated. METHODS: Experiments were conducted in large semi-field systems (SFS) with human volunteers inside, to mimic natural ecosystems, and using MLBs baited with natural or synthetic human odours and carbon dioxide. The MLBs were dusted with 10% pyriproxyfen (PPF) or entomopathogenic fungi (Metarhizium anisopliae) spores to mark mosquitoes physically contacting the devices. Each night, 400 laboratory-reared An. arabiensis females were released in one SFS chamber with two MLBs, and another chamber without MLBs (control). Mosquitoes were individually recaptured while attempting to bite volunteers inside SFS or by aspiration from SFS walls. Mosquitoes from chambers with PPF-treated MLBs and respective controls were individually dipped in water-filled cups containing ten conspecific third-instar larvae, whose subsequent development was monitored. Mosquitoes recaptured from chambers with fungi-treated MLBs were observed for fungal hyphal growth on their cadavers. Separately, effects on daily survival were determined by exposing An. arabiensis in chambers having MLBs treated with 5% pirimiphos methyl compared to chambers without MLBs (control), after which the mosquitoes were recaptured and monitored individually until they died. RESULTS: Up to 63% (152/240) and 43% (92/210) of mosquitoes recaptured inside treatment chambers were contaminated with pyriproxyfen and M. anisopliae, respectively, compared to 8% (19/240) and 0% (0/164) in controls. The mean number of larvae emerging from cups in which adults from chambers with PPF-treated MLBs were dipped was significantly lower [0.75 (0.50-1.01)], than in controls [28.79 (28.32-29.26)], P < 0.001). Daily survival of mosquitoes exposed to 5% pirimiphos methyl was nearly two-fold lower than controls [hazard ratio (HR) = 1.748 (1.551-1.920), P < 0.001]. CONCLUSION: High contamination rates in exposed mosquitoes even in presence of humans, demonstrates potential of MLBs for controlling outdoor-biting malaria vectors, either by reducing their survival or directly killing host-seeking mosquitoes. The MLBs also have potential for dispensing filial infanticides, such as PPF, which mosquitoes can transmit to their aquatic habitats for mosquito population control.

Predicting Scenarios for Successful Autodissemination of Pyriproxyfen by Malaria Vectors from Their Resting Sites to Aquatic Habitats; Description and Simulation Analysis of a Field-Parameterizable Model.

BACKGROUND: Large-cage experiments indicate pyriproxifen (PPF) can be transferred from resting sites to aquatic habitats by Anopheles arabiensis--malaria vector mosquitoes to inhibit emergence of their own offspring. PPF coverage is amplified twice: (1) partial coverage of resting sites with PPF contamination results in far higher contamination coverage of adult mosquitoes because they are mobile and use numerous resting sites per gonotrophic cycle, and (2) even greater contamination coverage of aquatic habitats results from accumulation of PPF from multiple oviposition events. METHODS AND FINDINGS: Deterministic mathematical models are described that use only field-measurable input parameters and capture the biological processes that mediate PPF autodissemination. Recent successes in large cages can be rationalized, and the plausibility of success under full field conditions can be evaluated a priori. The model also defines measurable properties of PPF delivery prototypes that may be optimized under controlled experimental conditions to maximize chances of success in full field trials. The most obvious flaw in this model is the endogenous relationship that inevitably occurs between the larval habitat coverage and the measured rate of oviposition into those habitats if the target mosquito species is used to mediate PPF transfer. However, this inconsistency also illustrates the potential advantages of using a different, non-target mosquito species for contamination at selected resting sites that shares the same aquatic habitats as the primary target. For autodissemination interventions to eliminate malaria transmission or vector populations during the dry season window of opportunity will require comprehensive contamination of the most challenging subset of aquatic habitats [Formula: see text] that persist or retain PPF activity (Ux) for only one week [Formula: see text], where Ux = 7 days). To achieve >99% contamination coverage of these habitats will necessitate values for the product of the proportional coverage of the ovipositing mosquito population with PPF contamination (CM) by the ovitrap-detectable rates of oviposition by wild mosquitoes into this subset of habitats [Formula: see text], divided by the titre of contaminated mosquitoes required to render them unproductive [Formula: see text], that approximately approach unity [Formula: see text]. CONCLUSIONS: The simple multiplicative relationship between CM and [Formula: see text], and the simple exponential decay effect they have upon uncontaminated aquatic habitats, allows application of this model by theoreticians and field biologists alike.

Increasing role of Anopheles funestus and Anopheles arabiensis in malaria transmission in the Kilombero Valley, Tanzania.

BACKGROUND: In order to sustain the gains achieved by current malaria control strategies, robust surveillance systems that monitor dynamics of vectors and their roles in malaria transmission over time are essential. This longitudinal study demonstrates the trends in malaria vector dynamics and their relative contribution to malaria transmission in hyperendemic transmission settings in Tanzania. METHODS: The study was conducted in two villages within the Kilombero Valley, in rural Tanzania for five consecutive years (2008-2012). Seventy-two houses were selected per village and each house was sampled for mosquitoes monthly using a CDC light trap. Collected mosquitoes were assessed for species identity and sporozoite infection status using PCR and ELISA, respectively. Anopheles funestus and Anopheles arabiensis susceptibility to insecticides was assessed using WHO guidelines. RESULTS: A total of 100,810 malaria vectors were collected, of which 76% were Anopheles gambiae s. l. and 24% were An. funestus. Of all An. funestus samples that amplified with PCR (n = 2,737), 97% were An. funestus s.s., 2% were Anopheles rivorulum and 1% Anopheles leesoni. Whereas for An. gambiae s.l. (n = 8,117), 93% were An. arabiensis and 7% were Anopheles gambiae s.s. The proportion of An. gambiae s.s. identified by PCR (2,924) declined from 0.2% in the year 2008 to undetectable levels in 2012. Malaria transmission intensity significantly decreased from an EIR of 78.14 infectious bites/person/year in 2008 to 35 ib/p/yr in 2011 but rebounded to 226 ib/p/yr in 2012 coinciding with an increased role of An. funestus in malaria transmission. Insecticide susceptibility tests indicated high levels of resistance in An. funestus against deltamethrin (87%), permethrin (65%), lambda cyhalothrin (74%), bendiocarb (65%), and DDT (66%). Similarly, An. arabiensis showed insecticide resistance to deltamethrin (64%), permethrin (77%) and lambda cyhalothrin (42%) in 2014. CONCLUSION: The results indicate the continuing role of An. arabiensis and the increasing importance of An. funestus in malaria transmission, and pyrethroid resistance development in both species. Complementary vector control and surveillance tools are needed that target the ecology, behaviour and insecticide resistance management of these vector species, in order to preserve the efficacy of LLINs.