A theoretical model for host-controlled regulation of symbiont density.

There is growing empirical evidence that animal hosts actively control the density of their mutualistic symbionts according to their requirements. Such active regulation can be facilitated by compartmentalization of symbionts within host tissues, which confers a high degree of control of the symbiosis to the host. Here, we build a general theoretical framework to predict the underlying ecological drivers and evolutionary consequences of host-controlled endosymbiont density regulation for a mutually obligate association between a host and a compartmentalized, vertically transmitted symbiont. Building on the assumption that the costs and benefits of hosting a symbiont population increase with symbiont density, we use state-dependent dynamic programming to determine an optimal strategy for the host, i.e., that which maximizes host fitness, when regulating the density of symbionts. Simulations of active host-controlled regulation governed by the optimal strategy predict that the density of the symbiont should converge to a constant level during host development, and following perturbation. However, a similar trend also emerges from alternative strategies of symbiont regulation. The strategy which maximizes host fitness also promotes symbiont fitness compared to alternative strategies, suggesting that active host-controlled regulation of symbiont density could be adaptive for the symbiont as well as the host. Adaptation of the framework allowed the dynamics of symbiont density to be predicted for other host-symbiont ecologies, such as for non-essential symbionts, demonstrating the versatility of this modelling approach.

Larval diet and temperature alter mosquito immunity and development: using body size and developmental traits to track carry-over effects on longevity.

BACKGROUND: Estimating arbovirus transmission potential requires a mechanistic understanding of how environmental factors influence the expression of adult mosquito traits. While preimaginal exposure to environmental factors can have profound effects on adult traits, tracking and predicting these effects remains challenging. METHODS: Using Aedes albopictus and a structural equation modeling approach, we explored how larval nutrition and temperature jointly affect development rate and success, female body size, and whether these metrics capture carry-over effects on adult female longevity. Additionally, we investigated how larval diet and temperature affect the baseline expression of 10 immune genes. RESULTS: We found that larval development success was primarily determined by diet, while temperature and diet both affected development rate and female body size. Under a low larval diet, pupal wet weight and wing length both declined with increasing temperature. In contrast, responses of the two morphometric measures to rearing temperature diverged when females were provided higher larval nutrition, with pupal wet weight increasing and wing length decreasing at higher temperatures. Our analyses also revealed opposing relationships between adult female lifespan and the two morphometric measures, with wing length having a positive association with longevity and pupal weight a negative association. Larval diet indirectly affected adult longevity, and the time to pupation was negatively correlated with longevity. The expression of eight immune genes from the toll, JAK-STAT and Imd pathways was enhanced in mosquitoes with higher nutrition. CONCLUSIONS: Our results highlight deficiencies from using a single body size measure to capture carry-over effects on adult traits. Further studies of larval development rate under varying environmental conditions and its potential for tracking carry-over effects on vectorial capacity are warranted.

Efficacy of a 'lethal house lure' against Culex quinquefasciatus from Bouaké city, Côte d'Ivoire.

BACKGROUND: Eave tube technology is a novel method of insecticide application that uses an electrostatic coating system to boost insecticide efficacy against resistant mosquitoes. A series of previous experiments showed encouraging insecticidal effects against malaria vectors. This study was undertaken to assess the effects of the eave tube approach on other Culicidae, in particular Culex quinquefasciatus, under laboratory and semi-field conditions. METHODS: Larvae of Cx. quinquefasciatus from Bouaké were collected and reared to adult stage, and World Health Organization (WHO) cylinder tests were performed to determine their resistance status. WHO standard 3-min cone bioassays were conducted using PermaNet 2.0 netting versus eave tube-treated inserts. To assess the transient exposure effect on Cx. quinquefasciatus, eave tube assay utilizing smelly socks as attractant was performed with exposure time of 30 s, 1 min, and 2 min on 10% beta-cyfluthrin-treated inserts. Residual activity of these treated inserts was then monitored over 9 months. Field tests involving release-recapture of Cx. quinquefasciatus within enclosures around experimental huts fitted with windows and untreated or insecticide-treated eave tubes were conducted to determine house entry preference and the impact of tubes on the survival of this species. RESULTS: Bouaké Cx. quinquefasciatus displayed high resistance to three out of four classes of insecticides currently used in public health. After 3 min of exposure in cone tests, 10% beta-cyfluthrin-treated inserts induced 100% mortality in Cx. quinquefasciatus, whereas the long-lasting insecticidal net (LLIN) only killed 4.5%. With reduced exposure time on the eave tube insert, mortality was still 100% after 2 min, 88% after 1 min, and 44% after 30 s. Mortality following 1 h exposure on 10% beta-cyfluthrin-treated insert was > 80% continuously up to 7 months post-treatment. Data suggest that Cx. quinquefasciatus have a stronger preference for entering a house through the eaves than through windows. Beta-cyfluthrin-treated inserts were able to kill 51% of resistant Cx. quinquefasciatus released within the enclosure. CONCLUSIONS: Eave tubes are a novel method for delivery of insecticide to the house. They attract nuisance host-seeking Cx. quinquefasciatus mosquitoes and are as effective in controlling them as they are against pyrethroid-resistant Anopheles gambiae, despite the high level of resistance Cx. quinquefasciatus have developed.

Exploring alternative insecticide delivery options in a "lethal house lure" for malaria vector control.

The In2Care EaveTube is a house modification designed to block and kill malaria mosquitoes using an electrostatic netting treated with insecticide powder. A previous study demonstrated prolonged duration of effective action of insecticide-treated electrostatic netting in a semi-field setting. As part of a cluster randomized controlled trial (CRT) of the EaveTube intervention in Côte d'Ivoire, we investigated the residual efficacy of a pyrethroid insecticide deployed in EaveTubes under village conditions of use. We also explored the scope of using existing malaria control technologies including LLINs and IRS as alternative methods to deliver insecticides in the lethal house lure. The efficacy of beta-cyfluthrin was monitored over time using the "eave tube bioassay" method. Mortality of beta-cyfluthrin exposed pyrethroid resistant Anopheles gambiae mosquitoes was > 80% after 4 months. The impact (mosquito mortality) of PVC tubes coated with pirimiphos methyl was similar to that of beta-cyfluthrin treated insert (66.8 vs. 62.8%) in release-recapture experiments in experimental huts. Efficacy was significantly lower with all the LLINs tested; however, the roof of PermaNet 3.0 induced significantly higher mosquito mortality (50.4%) compared to Olyset Plus (25.9%) and Interceptor G2 (21.6%) LLINs. The efficacy of the alternative delivery methods was short-lived with mortality decreasing below 50% within 2 months in residual activity bioassays. None of the products tested appeared superior to the powder treatments. Further research is therefore required to identify suitable insecticide delivery options in EaveTube for malaria vector control.

Incorporating effects of age on energy dynamics predicts nonlinear maternal allocation patterns in iteroparous animals.

Iteroparous parents face a trade-off between allocating current resources to reproduction versus maximizing survival to produce further offspring. Parental allocation varies across age and follows a hump-shaped pattern across diverse taxa, including mammals, birds and invertebrates. This nonlinear allocation pattern lacks a general theoretical explanation, potentially because most studies focus on offspring number rather than quality and do not incorporate uncertainty or age-dependence in energy intake or costs. Here, we develop a life-history model of maternal allocation in iteroparous animals. We identify the optimal allocation strategy in response to stochasticity when energetic costs, feeding success, energy intake and environmentally driven mortality risk are age-dependent. As a case study, we use tsetse, a viviparous insect that produces one offspring per reproductive attempt and relies on an uncertain food supply of vertebrate blood. Diverse scenarios generate a hump-shaped allocation when energetic costs and energy intake increase with age and also when energy intake decreases and energetic costs increase or decrease. Feeding success and environmentally driven mortality risk have little influence on age-dependence in allocation. We conclude that ubiquitous evidence for age-dependence in these influential traits can explain the prevalence of nonlinear maternal allocation across diverse taxonomic groups.

Insect-host control of obligate, intracellular symbiont density.

Many insects rely on intracellular bacterial symbionts to supplement their specialized diets with micronutrients. Using data from diverse and well-studied insect systems, we propose three lines of evidence suggesting that hosts have tight control over the density of their obligate, intracellular bacterial partners. First, empirical studies have demonstrated that the within-host symbiont density varies depending on the nutritional and developmental requirements of the host. Second, symbiont genomes are highly reduced and have limited capacity for self-replication or transcriptional regulation. Third, several mechanisms exist for hosts to tolerate, regulate and remove symbionts including physical compartmentalization and autophagy. We then consider whether such regulation is adaptive, by discussing the relationship between symbiont density and host fitness. We discuss current limitations of empirical studies for exploring fitness effects in host-symbiont relationships, and emphasize the potential for using mathematical models to formalize evolutionary hypotheses and to generate testable predictions for future work.

Effects of maternal age and stress on offspring quality in a viviparous fly.

Many organisms show signs of deterioration with age in terms of survival and reproduction. We tested whether intraspecific variation in such senescence patterns can be driven by resource availability or reproductive history. We did this by manipulating nutritional stress and age at first reproduction and measuring age-dependent reproductive output in tsetse (Glossina morsitans morsitans), a viviparous fly with high maternal allocation. Across all treatments, offspring weight followed a bell-shaped curve with maternal age. Nutritionally stressed females had a higher probability of abortion and produced offspring with lower starvation tolerance. There was no evidence of an increased rate of reproductive senescence in nutritionally stressed females, or a reduced rate due to delayed mating, as measured by patterns of abortion, offspring weight or offspring starvation tolerance. Therefore, although we found evidence of reproductive senescence in tsetse, our results did not indicate that resource allocation trade-offs or costs of reproduction increase the rate of senescence.

The role of human and mosquito behaviour in the efficacy of a house-based intervention.

Housing improvement such as blocking eaves and screening windows can help in reducing exposure to indoor biting mosquitoes. The impacts of physical barriers could potentially be boosted by the addition of a mechanism that kills mosquitoes as they attempt to enter the house. One example is to combine household screening with EaveTubes, which are insecticide-treated tubes inserted into closed eaves that attract and kill host-searching mosquitoes. The epidemiological impact of screening + EaveTubes is being evaluated in a large cluster randomized trial in Cote d'Ivoire. The study presented here is designed as a complement to this trial to help better understand the functional roles of screening and EaveTubes. We began by evaluating householder behaviour and household condition in the study villages. This work revealed that doors (and to some extent windows) were left open for large parts of the evening and morning, and that even houses modified to make them more 'mosquito proof' often had possible entry points for mosquitoes. We next built two realistic experimental houses in a village to enable us to explore how these aspects of behaviour and household quality affected the impact of screening and EaveTubes. We found that screening could have a substantial impact on indoor mosquito densities, even with realistic household condition and behaviour. By contrast, EaveTubes had no significant impact on indoor mosquito density, either as a stand-alone intervention or in combination with screening. However, there was evidence that mosquitoes recruited to the EaveTubes, and the resulting mortality could create a community benefit. These complementary modes of action of screening and EaveTubes support the rationale of combining the technologies to create a 'Lethal House Lure'. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Big Baby, Little Mother: Tsetse Flies Are Exceptions to the Juvenile Small Size Principle.

While across the animal kingdom offspring are born smaller than their parents, notable exceptions exist. Several dipteran species belonging to the Hippoboscoidea superfamily can produce offspring larger than themselves. In this essay, the blood-feeding tsetse is focused on. It is suggested that the extreme reproductive strategy of this fly is enabled by feeding solely on highly nutritious blood, and producing larval offspring that are soft and malleable. This immense reproductive expenditure may have evolved to avoid competition with other biting flies. Tsetse also transmit blood-borne parasites that cause the fatal diseases called African trypanosomiases. It is discussed how tsetse life history and reproductive strategy profoundly influence the type of vector control interventions used to reduce fly populations. In closing, it is argued that the unusual life history of tsetse warrants their preservation in the areas where human and animal health is not threatened.

Fine scale spatial investigation of multiple insecticide resistance and underlying target-site and metabolic mechanisms in Anopheles gambiae in central Côte d'Ivoire.

Routine monitoring of occurrence, levels and mechanisms of insecticide resistance informs effective management strategies, and should be used to assess the effect of new tools on resistance. As part of a cluster randomised controlled trial evaluating a novel insecticide-based intervention in central Côte d'Ivoire, we assessed resistance and its underlying mechanisms in Anopheles gambiae populations from a subset of trial villages. Resistance to multiple insecticides in An. gambiae s.s. and An. coluzzii was detected across villages, with dose-response assays demonstrating extremely high resistance intensity to the pyrethroid deltamethrin (> 1,500-fold), and mortality following exposure to pyrethroid-treated bednets was low (< 30% mortality in cone bioassays). The 1014F kdr mutation was almost fixed (≥ 90%) in all villages but the 1575Y kdr-amplifying mutation was relatively rare (< 15%). The carbamate and organophosphate resistance-associated Ace-1 G119S mutation was also detected at moderate frequencies (22-43%). Transcriptome analysis identified overexpression of P450 genes known to confer pyrethroid resistance (Cyp9K1, Cyp6P3, and Cyp6M2), and also a carboxylesterase (COEAE1F) as major candidates. Cyp6P3 expression was high but variable (up to 33-fold) and correlated positively with deltamethrin resistance intensity across villages (r2 = 0.78, P = 0.02). Tools and strategies to mitigate the extreme and multiple resistance provided by these mechanisms are required in this area to avoid future control failures.

Semi-field evaluation of the cumulative effects of a "Lethal House Lure" on malaria mosquito mortality.

BACKGROUND: There is growing interest in the potential to modify houses to target mosquitoes with insecticides or repellents as they search for human hosts. One version of this 'Lethal House Lure' approach is the In2Care® EaveTube, which consists of a section of polyvinyl chloride (PVC) pipe fitted into a closed eave, with an insert comprising electrostatic netting treated with insecticide powder placed inside the tube. Preliminary evidence suggests that when combined with screening of doors and windows, there is a reduction in entry of mosquitoes and an increase in mortality. However, the rate of overnight mortality remains unclear. The current study used a field enclosure built around experimental huts to investigate the mortality of cohorts of mosquitoes over multiple nights. METHODS: Anopheles gambiae sensu lato mosquitoes were collected from the field as larvae and reared through to adult. Three-to-five days old adult females were released inside an enclosure housing two modified West African style experimental huts at a field site in M'be, Côte d'Ivoire. Huts were either equipped with insecticide-treated tubes at eave height and had closed windows (treatment) or had open windows and open tubes (controls). The number of host-seeking mosquitoes entering the huts and cumulative mortality were monitored over 2 or 4 days. RESULTS: Very few (0-0.4%) mosquitoes were able to enter huts fitted with insecticide-treated tubes and closed windows. In contrast, mosquitoes continually entered the control huts, with a cumulative mean of 50-80% over 2 to 4 days. Baseline mortality with control huts was approximately 2-4% per day, but the addition of insecticide-treated tubes increased mortality to around 25% per day. Overall cumulative mortality was estimated to be up to 87% over 4 days when huts were fitted with tubes. CONCLUSION: Only 20-25% of mosquitoes contacted insecticide-treated tubes or entered control huts in a given night. However, mosquitoes continue to host search over sequential nights, and this can lead to high cumulative mortality over 2 to 4 days. This mortality should contribute to community-level reduction in transmission assuming sufficient coverage of the intervention.

Screening and field performance of powder-formulated insecticides on eave tube inserts against pyrethroid resistant Anopheles gambiae s.l.: an investigation into 'actives' prior to a randomized controlled trial in Côte d'Ivoire.

BACKGROUND: The widespread emergence of insecticide resistance in African malaria vectors remains one of the main challenges facing control programmes. Electrostatic coating that uses polarity to bind insecticide particles is a new way of delivering insecticides to mosquitoes. Although previous tests demonstrated the resistance breaking potential of this application method, studies screening and investigating the residual efficacy of a broader range of insecticides are necessary. METHODS: Eleven insecticide powder formulations belonging to six insecticide classes (pyrethroid, carbamate, organophosphate, neonicotinoid, entomopathogenic fungus and boric acid) were initially screened for residual activity over 4 weeks against pyrethroid resistant Anopheles gambiae sensu lato (s.l.) from the M'bé valley, central Côte d'Ivoire. Tests were performed using the eave tube assay that simulates the behavioural interaction between mosquitoes and insecticide-treated inserts. With the best performing insecticide, persistence was monitored over 12 months and the actual contact time lethal to mosquitoes was explored, using a range of transient exposure time (5 s, 30 s, 1 min up to 2 min) in the tube assays in laboratory. The mortality data were calibrated against overnight release-recapture data from enclosure around experimental huts incorporating treated inserts at the M'bé site. The natural recruitment rate of mosquitoes to the tube without insecticide treatment was assessed using fluorescent dust particles. RESULTS: Although most insecticides assayed during the initial screening induced significant mortality (45-100%) of pyrethroid resistant An. gambiae during the first 2 weeks, only 10% beta-cyfluthrin retained high residual efficacy, killing 100% of An. gambiae during the first month and > 80% over 8 subsequent months. Transient exposure for 5 s of mosquitoes to 10% beta-cyfluthrin produced 56% mortality, with an increase to 98% when contact time was extended to 2 min (P = 0.001). In the experimental hut enclosures, mortality of An. gambiae with 10% beta-cyfluthrin treated inserts was 55% compared to similar rate (44%) of mosquitoes that contacted the inserts treated with fluorescent dusts. This suggests that all host-seeking female mosquitoes that contacted beta-cyfluthrin treated inserts during host-seeking were killed. CONCLUSION: The eave tube technology is a novel malaria control approach which combines house proofing and targeted control of anopheline mosquitoes using insecticide treated inserts. Beta-cyfluthrin showed great promise for providing prolonged control of pyrethroid resistant An. gambiae and has potential to be deployed year-round in areas where malaria parasites are transmitted by highly pyrethroid resistant An. gambiae across sub-Saharan Africa.

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.

Semi-field studies to better understand the impact of eave tubes on mosquito mortality and behaviour.

BACKGROUND: Eave tubes are a type of housing modification that provide a novel way of delivering insecticides to mosquitoes as they attempt to enter the house. The current study reports on a series of semi-field studies aimed at improving the understanding of how eave tubes might impact mosquito mortality and behaviour. METHODS: Experiments were conducted using West African style experimental huts at a field site in M'be, Côte d'Ivoire. Huts were modified in various ways to determine: (i) whether mosquitoes in this field setting naturally recruit to eave tubes; (ii) whether eave tubes can reduce house entry even in the absence of screening; (iii) whether mosquitoes suffer mortality if they attempt to exit a house via treated eave tubes; and, (iv) whether screening and eave tubes might deflect mosquitoes into neighbouring houses without the intervention. RESULTS: Ninety percent more mosquitoes (Anopheles gambiae sensu lato, and other species) entered huts through open eaves tubes compared to window slits. The addition of insecticide-treated eave tubes reduced mosquito entry by 60%, even when windows remained open. Those mosquitoes that managed to enter the huts exhibited a 64% reduction in blood feeding and a tendency for increased mortality, suggesting contact with insecticide-treated inserts prior to hut entry. When An. gambiae mosquitoes were deliberately introduced into huts with treated eave tubes, there was evidence of six times increase in overnight mortality, suggesting mosquitoes can contact treated eave tube inserts when trying to exit the hut. There was no evidence for deflection of mosquitoes from huts with screening, or screening plus eave tubes, to adjacent unmodified huts. CONCLUSIONS: Eave tubes are a potentially effective way to target Anopheles mosquitoes with insecticides. That treated eave tubes can reduce mosquito entry even when windows are open is a potentially important result as it suggests that eave tubes might not need to be combined with household screening to have an impact on malaria transmission. The absence of deflection is also a potentially important result as coverage of eave tubes and/or screening is unlikely to be 100% and it is important that households that do not have the technology are not disadvantaged by those that do.

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.