Video augmentation of the WHO cone assay to quantify mosquito behavioural responses to insecticide-treated nets.

BACKGROUND: Insecticide-treated nets (ITNs) using pyrethroids have been the main vector control tools deployed in malaria endemic countries and are responsible for the dramatic reduction in African malaria cases in the early 2000s. The World Health Organization (WHO) cone test was designed to assess the rapid toxicity effects of pyrethroid exposure on mosquito vectors but has yielded no insights beyond 60-min knockdown and 24-h mortality. As dual-active-ingredient (AI) ITNs become more widespread, bioassays that can provide realistic assessment of single- and dual-treated ITNs (i.e. nets with more than one active ingredient) are urgently needed. METHODS: We present an augmentation of the cone test that enables accurate quantification of vector behavioural responses (specifically movement, spatial and temporal occupancy) to ITNs using video recording and bespoke software that uses background segmentation methods to detect spatial changes in the movement of mosquitoes within the cone. Four strains of Anopheles gambiae sensu lato (s.l.) were exposed to four ITNs (PermaNet 2.0, PermaNet 3.0, Olyset Net, Interceptor G2) and untreated nets in these modified cone tests. Life history data (post-exposure blood-feeding, blood meal weight, longevity) for individual mosquitoes were recorded. RESULTS: All mosquitoes responded to the presence of ITNs, spending from 1.48 to 3.67 times more time in the upper region of the cone, depending on the ITN type. Of all ITNs, PermaNet 2.0 provoked the smallest change in behavioural response. Activity in the cone influenced observed post-exposure longevity, and in resistant strains exposed to Interceptor G2, the higher the activity, the greater the risk of dying, as long as the proportion of activity at the net surface was less than 50%. All ITNs inhibited blood-feeding, and smaller blood meals were taken when mosquitoes fed. CONCLUSIONS: The additional mosquito behaviour data obtained by using this modification to the WHO cone test provides unique insight into the innate responses of different mosquito strains on untreated nets and the entomological mode of action of ITNs, important evidence when evaluating ITN characteristics.

Pyrethroid-treated bed nets impair blood feeding performance in insecticide resistant mosquitoes.

The blood feeding performance of female mosquitoes directly impacts their ability to transmit malaria. Yet their host seeking and blood feeding behaviours in the presence of insecticide-treated nets (ITNs) are still poorly understood. This work explores how both insecticide resistant and susceptible Anopheles gambiae s.l. mosquitoes interact with pyrethroid nets (PermaNet 2.0 or Olyset net) or an untreated net (UTN) while attempting to blood feed on a human arm. Regardless of mosquito resistance status, the ITNs did not efficiently prevent host searching but reduced blood feeding success by 34.1 (29.31-38.95) %. The Permanet and Olyset net reduced to 227.5 (208.19-246.77) sec and 235.9 (214.03-257.74) sec the average blood feeding duration from 369.9 (342.78-397.04) sec with the UTN. The ingested blood volume was on average 22% lower for all mosquitoes exposed to insecticide. When feeding through ITNs, the blood volume flow rate of the susceptible strain increased by 35%, but no significant difference was found in the resistant strain. Thus, whilst the presence of the insecticide in ITNs reduced mosquito blood feeding success and blood volume, the mosquito's ability to respond by accelerating her rate of blood ingestion may further reduce the impact of ITNs on resistant mosquitoes.

Omitting age-dependent mosquito mortality in malaria models underestimates the effectiveness of insecticide-treated nets.

Mathematical models of vector-borne infections, including malaria, often assume age-independent mortality rates of vectors, despite evidence that many insects senesce. In this study we present survival data on insecticide-resistant Anopheles gambiae s.l. from experiments in Côte d'Ivoire. We fit a constant mortality function and two age-dependent functions (logistic and Gompertz) to the data from mosquitoes exposed (treated) and not exposed (control) to insecticide-treated nets (ITNs), to establish biologically realistic survival functions. This enables us to explore the effects of insecticide exposure on mosquito mortality rates, and the extent to which insecticide resistance might impact the effectiveness of ITNs. We investigate this by calculating the expected number of infectious bites a mosquito will take in its lifetime, and by extension the vectorial capacity. Our results show that the predicted vectorial capacity is substantially lower in mosquitoes exposed to ITNs, despite the mosquitoes in the experiment being highly insecticide-resistant. The more realistic age-dependent functions provide a better fit to the experimental data compared to a constant mortality function and, hence, influence the predicted impact of ITNs on malaria transmission potential. In models with age-independent mortality, there is a great reduction for the vectorial capacity under exposure compared to no exposure. However, the two age-dependent functions predicted an even larger reduction due to exposure, highlighting the impact of incorporating age in the mortality rates. These results further show that multiple exposures to ITNs had a considerable effect on the vectorial capacity. Overall, the study highlights the importance of including age dependency in mathematical models of vector-borne disease transmission and in fully understanding the impact of interventions.

Use of novel lab assays to examine the effect of pyrethroid-treated bed nets on blood-feeding success and longevity of highly insecticide-resistant Anopheles gambiae s.l. mosquitoes.

BACKGROUND: There is a pressing need to improve understanding of how insecticide resistance affects the functional performance of insecticide-treated nets (ITNs). Standard WHO insecticide resistance monitoring assays are designed for resistance surveillance and do not necessarily provide insight into how different frequencies, mechanisms or intensities of resistance affect the ability of ITNs to reduce malaria transmission. METHODS: The current study presents some novel laboratory-based assays that attempt to better simulate realistic exposure of mosquitoes to ITNs and to quantify impact of exposure not only on instantaneous mortality, but also on blood-feeding and longevity, two traits that are central to transmission. The assays evaluated the performance of a standard ITN (Permanet® 2.0; Vestergaard Frandsen), a 'next generation' combination ITN with a resistance-breaking synergist (Permanet® 3.0) and an untreated net (UTN), against field-derived Anopheles gambiae sensu lato mosquitoes from Côte d'Ivoire exhibiting a 1500-fold increase in pyrethroid resistance relative to a standard susceptible strain. RESULTS: The study revealed that the standard ITN induced negligible instantaneous mortality against the resistant mosquitoes, whereas the resistance-breaking net caused high mortality and a reduction in blood-feeding. However, both ITNs still impacted long-term survival relative to the UTN. The impact on longevity depended on feeding status, with blood-fed mosquitoes living longer than unfed mosquitoes following ITN exposure. Exposure to both ITNs also reduced the blood-feeding success, the time spent on the net and blood-feeding duration, relative to the untreated net. CONCLUSION: Although a standard ITN did not have as substantial instantaneous impact as the resistance-breaking net, it still had significant impacts on traits important for transmission. These results highlight the benefit of improved bioefficacy assays that allow for realistic exposure and consider sub- or pre-lethal effects to help assess the functional significance of insecticide resistance.

The relationship between size and longevity of the malaria vector Anopheles gambiae (s.s.) depends on the larval environment.

BACKGROUND: Understanding the variation in vector-borne disease transmission intensity across time and space relies on a thorough understanding of the impact of environmental factors on vectorial capacity traits of mosquito populations. This is driven primarily by variation in larval development and growth, with carryover effects influencing adult traits such as longevity and adult body size. The relationship between body size and longevity strongly affects the evolution of life histories and the epidemiology of vector-borne diseases. This relationship ranges from positive to negative but the reasons for this variability are not clear. Both traits depend on a number of environmental factors, but primarily on temperature as well as availability of nutritional resources. We therefore asked how the larval environment of the mosquito Anopheles gambiae Giles (sensu stricto) (Diptera: Culicidae) affects the relationship between body size and longevity. METHODS: We reared the larvae of An. gambiae individually at three temperatures (21, 25 and 29 °C) and two food levels (the standard and 50% of our laboratory diet) and measured adult size and longevity. We estimated the direct and indirect (via adult size) effects of food and temperature on longevity with a piecewise structural equation model (SEM). RESULTS: We confirmed the direct effects of food and temperature during larval development on body size, as wing length decreased with increasing temperature and decreasing food levels. While the overall relationship between size and longevity was weak, we measured striking differences among environments. At 25 °C there was no clear relationship between size and longevity; at 29 °C the association was negative with standard food but positive with low food; whereas at 21 °C it was positive with standard food but negative with low food. CONCLUSIONS: The larval environment influences the adult's fitness in complex ways with larger mosquitoes living longer in some environments but not in others. This confirmed our hypothesis that the relationship between size and longevity is not limited to a positive correlation. A better understanding of this relationship and its mechanisms may improve the modelling of the transmission of vector borne diseases, the evolution of life history traits, and the influence of vector control.

Rethinking the extrinsic incubation period of malaria parasites.

The time it takes for malaria parasites to develop within a mosquito, and become transmissible, is known as the extrinsic incubation period, or EIP. EIP is a key parameter influencing transmission intensity as it combines with mosquito mortality rate and competence to determine the number of mosquitoes that ultimately become infectious. In spite of its epidemiological significance, data on EIP are scant. Current approaches to estimate EIP are largely based on temperature-dependent models developed from data collected on parasite development within a single mosquito species in the 1930s. These models assume that the only factor affecting EIP is mean environmental temperature. Here, we review evidence to suggest that in addition to mean temperature, EIP is likely influenced by genetic diversity of the vector, diversity of the parasite, and variation in a range of biotic and abiotic factors that affect mosquito condition. We further demonstrate that the classic approach of measuring EIP as the time at which mosquitoes first become infectious likely misrepresents EIP for a mosquito population. We argue for a better understanding of EIP to improve models of transmission, refine predictions of the possible impacts of climate change, and determine the potential evolutionary responses of malaria parasites to current and future mosquito control tools.

Priorities for Broadening the Malaria Vector Control Tool Kit.

Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) have contributed substantially to reductions in the burden of malaria in the past 15 years. Building on this foundation, the goal is now to drive malaria towards elimination. Vector control remains central to this goal, but there are limitations to what is achievable with the current tools. Here we highlight how a broader appreciation of adult mosquito behavior is yielding a number of supplementary approaches to bolster the vector-control tool kit. We emphasize tools that offer new modes of control and could realistically contribute to operational control in the next 5 years. Promoting complementary tools that are close to field-ready is a priority for achieving the global malaria-control targets.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response.

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Larval environment influences vector competence of the malaria mosquito Anopheles gambiae.

Background: While environmental factors such as temperature can influence the vector competence of mosquitoes directly, for example by affecting the longevity of the mosquito and the development of the malaria parasite they may also have an indirect impact on the parasite's transmission. By influencing larval development, they may affect the adult traits that are important for the parasite's development and transmission. We studied the influence of two larval environmental factors, food availability and temperature, on the probability that mosquitoes infected with the malaria parasite survived to harbour sporozoites in their salivary glands. Materials and methods: Anopheles gambiae larvae were reared at 21ºC, 25ºC or 29ºC, and fed either a standard larval diet or half of it. Adults could blood feed on mice harbouring the infectious gametocytic stage of Plasmodium berghei ANKA transformed with green fluorescent protein (GFP). Survival was assessed every 24 hrs up to 21 days post infection, when surviving mosquitoes were dissected to check the salivary glands for sporozoites with a fluorescent microscope sensitive to GFP. Using a binomial GLM we analysed 'vector competence', i.e. if mosquitoes survived until dissection and harboured sporozoites in their salivary glands. Results: Vector competence dropped by about a third if we fed larvae half the standard food regime. The effect of temperature during the larval period depended strongly on the food regime. At low food, increasing temperature from 21ºC to 29ºC increased vector competence from about 0.18 to 0.48, whereas at standard food, vector competence dropped from about 0.67 at 21ºC to 0.56 at 29ºC. Conclusions: Thus, perceptions and models about the role of environmental change on the transmission of malaria should include how the environment changes adult life-history by influencing larval development.