Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs).

BACKGROUND: "Regeneration time" (RT) denotes the time required to obtain a stable mortality rate for mosquitoes exposed to insecticide-treated nets (ITNs) after three consecutive washes of a net in a day. The RT informs the wash interval used to artificially age ITNs to simulate their lifetime performance under user conditions (20 washes). RT was estimated following World Health Organization (WHO) longitudinal method (LM) procedures. Longitudinal evaluation may introduce heterogeneity due to mosquito batch variability, complicating RT determination. To overcome this, nets at each stage of regeneration (i.e., 1, 2, 3, 5 and 7 days post wash) were prepared in advance and refrigerated; then, a complete regeneration series was tested with a single mosquito batch on 1 testing day, completing four series over 4 days. This study compared the complete series method (CSM) against the LM. METHODS: The overall heterogeneity in the methods for estimating RT of one incorporated alpha-cypermethrin and piperonyl butoxide (PBO) and one incorporated permethrin with PBO ITNs was determined using laboratory-reared resistant Anopheles arabiensis under standard laboratory conditions. LM methods and CSM were compared in two experiments with refrigerated nets acclimated for (i) 2 h (test 1) and (ii) 3 h (test 2). Four regeneration replicates per day were tested per ITN product with 50 mosquitoes exposed per replicate (equivalent sample size to LM). The heterogeneity from these methods was compared descriptively. RESULTS: The intra-method variability for unwashed pieces was minimal, with variance of 1.26 for CSM and 1.18 for LM. For unwashed nets, LM had substantially greater variance and ratio of LM:CSM was 2.66 in test 1 and 2.49 in test 2. The magnitude of mortality measured in bioassays depended on sample acclimation after refrigeration. CONCLUSIONS: The CSM is a convenient method for determining the regeneration times. ITNs are prepared in advance, reducing pressure to prepare all samples to start on a single day. A complete regeneration series of samples is removed from the refrigerator, defrosted and evaluated on a single day with one mosquito batch reducing the influence of mosquito batch heterogeneity on results. Replicates can be conducted over several days but do not have to be conducted on consecutive days, allowing easy facility scheduling.

Sugar and blood: the nutritional priorities of the dengue vector, Aedes aegypti.

BACKGROUND: Sugar-feeding behaviour is essential for mosquito survival and reproduction, and has been exploited to develop new control strategies, such as the attractive targeted sugar baits (ATSB). This study examined the sugar-feeding habits of the dengue vector, Aedes aegypti, in semi-field conditions to determine the optimal timing (age) of sugar meals and whether the availability of sugar sources could affect blood-feeding by these mosquitoes. METHODS: A series of paired-choice assays were conducted in which mosquitoes were allowed to choose between a sugar meal or a blood meal directly from a rabbit. Female 1-day-old mosquitoes were given meal choices in cages I-V and observed for feeding choice in only one cage every day for 5 days starting with cages I to V. The preference of Ae. aegypti to feed on sugar or blood and the effect of sugar source availability on blood-feeding was assessed at different chronological and physiological ages. RESULTS: In the first 5 days post-emergence, there was no significant difference in mosquito preference for sugar or blood meals. However, after the first gonotrophic cycle, they had a greater preference for blood over sugar (odds ratio, OR [95% confidence interval, CI] = 9.4 [6.7-13.0]; P < 0.001). Nulliparous Ae. aegypti females (≤ 5-day-old mosquitoes) were less likely to blood-feed if both sugar and blood sources were concurrently available (OR = 0.06 [0.02-0.16]; P < 0.001). CONCLUSIONS: Newly emerged females of Ae. aegypti mosquitoes were equally likely to choose a sugar meal or a blood meal. However, after the first gonotrophic cycle, they had a greater preference for blood over sugar. Additionally, nulliparous female mosquitoes were less likely to blood-feed when both sugar and blood sources were available. These findings provide insights into the sugar-feeding behaviour of Ae. aegypti and can inform the development and optimization of new control strategies such as using ATSB.

Life expectancy of Anopheles funestus is double that of Anopheles arabiensis in southeast Tanzania based on mark-release-recapture method.

Anopheles arabiensis and Anopheles funestus sensu stricto mosquitoes are major East African malaria vectors. Understanding their dispersal and population structure is critical for developing effective malaria control tools. Three mark-release-recapture (MRR) experiments were conducted for 51 nights to assess daily survival and flight range of An. arabiensis and An. funestus mosquitoes in south-eastern, Tanzania. Mosquitoes were marked with a fluorescent dye as they emerged from breeding sites via a self-marking device. Mosquitoes were collected indoors and outdoors using human landing catches (HLC) and Centers for Disease Control and Prevention light traps (CDC-LT). In total, 4210 An. arabiensis and An. funestus were collected with 316 (7.5%) marked and recaptured (MR). Daily mean MR was 6.8, standard deviation (SD ± 7.6) for An. arabiensis and 8.9 (SD ± 8.3) for An. funestus. Probability of daily survival was 0.76 for An. arabiensis and 0.86 for An. funestus translating into average life expectancy of 3.6 days for An. arabiensis and 6.5 days for An. funestus. Dispersal distance was 654 m for An. arabiensis and 510 m for An. funestus. An. funestus life expectancy was substantially longer than that of An. arabiensis. The MRR method described here could be routinely utilized when evaluating the impact of new vector control tools on mosquito survival.

Efficacy of the spatial repellent product Mosquito Shield™ against wild pyrethroid-resistant Anopheles arabiensis in south-eastern Tanzania.

BACKGROUND: Spatial repellents that create airborne concentrations of an active ingredient (AI) within a space offer a scalable solution to further reduce transmission of malaria, by disrupting mosquito behaviours in ways that ultimately lead to reduced human-vector contact. Passive emanator spatial repellents can protect multiple people within the treated space and can last for multiple weeks without the need for daily user touchpoints, making them less intrusive interventions. They may be particularly advantageous in certain use cases where implementation of core tools may be constrained, such as in humanitarian emergencies and among mobile at-risk populations. The purpose of this study was to assess the efficacy of Mosquito Shield™ deployed in experimental huts against wild, free-flying, pyrethroid-resistant Anopheles arabiensis mosquitoes in Tanzania over 1 month. METHODS: The efficacy of Mosquito Shield™ transfluthrin spatial repellent in reducing mosquito lands and blood-feeding was evaluated using 24 huts: sixteen huts were allocated to Human Landing Catch (HLC) collections and eight huts to estimating blood-feeding. In both experiments, half of the huts received no intervention (control) while the remaining received the intervention randomly allocated to huts and remained fixed for the study duration. Outcomes measured were mosquito landings, blood-fed, resting and dead mosquitoes. Data were analysed by multilevel mixed effects regression with appropriate dispersion and link function accounting for volunteer, hut and day. RESULTS: Landing inhibition was estimated to be 70% (57-78%) [IRR 0.30 (95% CI 0.22-0.43); p < 0.0001] and blood-feeding inhibition was estimated to be 69% (56-79%) [IRR 0.31 (95% CI 0.21-0.44; p < 0.0001] There was no difference in the protective efficacy estimates of landing and blood-feeding inhibition [IRR 0.98 (95% CI 0.53-1.82; p = 0.958]. CONCLUSIONS: This study demonstrated that Mosquito Shield™ was efficacious against a wild pyrethroid-resistant strain of An. arabiensis mosquitoes in Tanzania for up to 1 month and could be used as a complementary or stand-alone tool where gaps in protection offered by core malaria vector control tools exist. HLC is a suitable technique for estimating bite reductions conferred by spatial repellents especially where direct blood-feeding measurements are not practical or are ethically limited.

CDC light traps underestimate the protective efficacy of an indoor spatial repellent against bites from wild Anopheles arabiensis mosquitoes in Tanzania.

BACKGROUND: Methods for evaluating efficacy of core malaria interventions in experimental and operational settings are well established but gaps exist for spatial repellents (SR). The objective of this study was to compare three different techniques: (1) collection of blood-fed mosquitoes (feeding), (2) human landing catch (HLC), and (3) CDC light trap (CDC-LT) collections for measuring the indoor protective efficacy (PE) of the volatile pyrethroid SR product Mosquito Shield™ METHODS: The PE of Mosquito Shield™ against a wild population of pyrethroid-resistant Anopheles arabiensis mosquitoes was determined via feeding, HLC, or CDC-LT using four simultaneous 3 by 3 Latin squares (LS) run using 12 experimental huts in Tanzania. On any given night each technique was assigned to two huts with control and two huts with treatment. The LS were run twice over 18 nights to give a sample size of 72 replicates for each technique. Data were analysed by negative binomial regression. RESULTS: The PE of Mosquito Shield™ measured as feeding inhibition was 84% (95% confidence interval (CI) 58-94% [Incidence Rate Ratio (IRR) 0.16 (0.06-0.42), p < 0.001]; landing inhibition 77% [64-86%, (IRR 0.23 (0.14-0.36) p < 0.001]; and reduction in numbers collected by CDC-LT 30% (0-56%) [IRR 0.70 (0.44-1.0) p = 0.160]. Analysis of the agreement of the PE measured by each technique relative to HLC indicated no statistical difference in PE measured by feeding inhibition and landing inhibition [IRR 0.73 (0.25-2.12) p = 0.568], but a significant difference in PE measured by CDC-LT and landing inhibition [IRR 3.13 (1.57-6.26) p = 0.001]. CONCLUSION: HLC gave a similar estimate of PE of Mosquito Shield™ against An. arabiensis mosquitoes when compared to measuring blood-feeding directly, while CDC-LT underestimated PE relative to the other techniques. The results of this study indicate that CDC-LT could not effectively estimate PE of the indoor spatial repellent in this setting. It is critical to first evaluate the use of CDC-LT (and other tools) in local settings prior to their use in entomological studies when evaluating the impact of indoor SR to ensure that they reflect the true PE of the intervention.

Evaluation of an ivermectin-based attractive targeted sugar bait (ATSB) against Aedes aegypti in Tanzania.

Background The control of vector borne arboviral diseases such as Dengue is mainly achieved by reducing human-vector contact and controlling the vectors through source reduction and environmental management. These measures are constrained by labour intensity, insecticide resistance and pro-active community participation. The current study intended to develop and test an ivermectin-based attractive-targeted sugar bait (ATSB) against Aedes aegypti. Methods The 48hour lethal concentration (LC90) of ivermectin against Ae. aegypti was determined through serial dilution experiment where five 30cm x 30cm x 30cm cages were set; into each, a 10% sugar solution treated with ivermectin were introduced. 40 Ae. aegypti were released into each cage and observed for mortality after 4, 8, 24 and 48 hours. The ivermectin-based ATSB was evaluated in a semi field system where ATSB and attractive sugar bait (ASB) were deployed into each compartment of the semi field and 100 female Ae. aegypti were released every day and recaptured the next day through human land catch and Bio-gent sentinel trap. The developed and semi-field tested ATSB was further tested in the field by deploying them in garages. Results The ivermectin 48hr LC90 of male and female Ae. aegypti was found to be 0.03% w/v. In the semi field system, the ATSB significantly reduced a free-flying population of Ae. aegypti within 24 hours (incidence rate ratio (IRR) = 0.62; [95% confidence interval (95%CI); 0.54-0.70] and p-value < 0.001). However, in the field, the ATSBs required the addition of yeast as a carbon dioxide source to efficiently attract Ae. aegypti mosquitoes to feed. Conclusion Ivermectin is an active ingredient that can be used in an ATSB for Ae. aegypti depopulation. However, further research is needed to improve the developed and tested ATSB to compete with natural sources of sugar in a natural environment.