Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development

One potential strategy for the control of malaria and other vector-borne diseases is the introduction into wild vector populations of genetic constructs that reduce vectorial capacity. An important caveat of this approach is that the genetic construct should have minimal fitness cost to the transformed vector. Previously, we produced transgenic Anopheles stephensi expressing either of two effector genes, a tetramer of the SM1 dodecapeptide or the phospholipase A2 gene (PLA2) from honeybee venom. Mosquitoes carrying either of these transgenes were impaired for Plasmodium berghei transmission. We have investigated the role of two effector genes for malaria parasite blockage in terms of the fitness imposed to the mosquito vector that expresses either molecule. By measuring mosquito survival, fecundity, fertility, and by running population cage experiments, we found that mosquitoes transformed with the SM1 construct showed no significant reduction in these fitness parameters relative to nontransgenic controls. The PLA2 transgenics, however, had reduced fitness that seemed to be independent of the insertion site of the transgene. We conclude that the fitness load imposed by refractory gene(s)-expressing mosquitoes depends on the effect of the transgenic protein produced in that mosquito. These results have important implications for implementation of malaria control via genetic modification of mosquitoes.     

Linking individual phenotype to density-dependent population growth: the influence of body size on the population dynamics of malaria vectors

Understanding the endogenous factors that drive the population dynamics of malaria mosquitoes will facilitate more accurate predictions about vector control effectiveness and our ability to destabilize the growth of either low- or high-density insect populations. We assessed whether variation in phenotypic traits predict the dynamics of Anopheles gambiae sensu lato mosquitoes, the most important vectors of human malaria. Anopheles gambiae dynamics were monitored over a six-month period of seasonal growth and decline. The population exhibited density-dependent feedback, with the carrying capacity being modified by rainfall (97% wAIC(c) support). The individual phenotypic expression of the maternal (p = 0.0001) and current (p = 0.040) body size positively influenced population growth. Our field-based evidence uniquely demonstrates that individual fitness can have population-level impacts and, furthermore, can mitigate the impact of exogenous drivers (e.g. rainfall) in species whose reproduction depends upon it. Once frontline interventions have suppressed mosquito densities, attempts to eliminate malaria with supplementary vector control tools may be attenuated by increased population growth and individual fitness.     

Mosquito mortality and the evolution of malaria virulence

Abstract Several laboratory studies of malaria parasites (Plasmodium sp.) and some field observations suggest that parasite virulence, defined as the harm a parasite causes to its vertebrate host, is positively correlated with transmission. Given this advantage, what limits the continual evolution of higher parasite virulence? One possibility is that while more virulent strains are more infectious, they are also more lethal to mosquitoes. In this study, we tested whether the virulence of the rodent malaria parasite P. chabaudi in the laboratory mouse was correlated with the fitness of mosquitoes it subsequently infected. Mice were infected with one of seven genetically distinct clones of P. chabaudi that differ in virulence. Weight loss and anemia in infected mice were monitored for 16–17 days before Anopheles stephensi mosquitoes were allowed to take a blood meal from them. Infection virulence in mice was positively correlated with transmission to mosquitoes (infection rate) and weakly associated with parasite burden (number of oocysts). Mosquito survival fell with increasing oocyst burden, but there was no overall statistically significant relationship between virulence in mice and mosquito mortality. Thus, there was no evidence that more virulent strains are more lethal to mosquitoes. Both vector survival and fecundity depended on parasite clone, and contrary to expectations, mosquitoes fed on infections more virulent to mice were more fecund. The strong parasite genetic effects associated with both fecundity and survival suggests that vector fitness could be an important selective agent shaping malaria population genetics and the evolution of phenotypes such as virulence in the vector.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Dynamics of malaria transmission model with sterile mosquitoes

In this paper, a malaria transmission model with sterile mosquitoes is considered. We first formulate a simple SEIR malaria transmission model as our baseline model. Then sterile mosquitoes are introduced into the baseline model. We consider the case that the release rate of sterile mosquitoes is proportional to the wild mosquito population size. To investigate the impact of releasing sterile mosquitoes on the malaria transmission, the dynamics of the baseline model and the models with the sterile mosquitoes are discussed. We derive formulas of the reproductive numbers and explore the existence of endemic equilibrium as the reproductive number is more than unity for these models. It is shown that both the baseline model and the models with the sterile mosquitoes undergo backward bifurcations. Based on theoretical analysis and numerical simulation, we investigate the impact of releasing sterile mosquitoes on malaria transmission.     

Using evolutionary costs to enhance the efficacy of malaria control via genetically manipulated mosquitoes

An earlier mathematical model exploring the use of genetically manipulated mosquitoes for malaria control suggested that the prevalence of malaria is reduced significantly only if almost all mosquitoes become completely resistant to malaria. Central to the model was the 'cost of resistance': the reduction of a resistant mosquito's evolutionary fitness in comparison with a sensitive one's. Here, we consider the possibility of obtaining more optimistic outcomes by taking into account the epidemiological (in addition to the evolutionary) consequences of a cost of resistance that decreases the life-span of adult mosquitoes (the most relevant parameter for the parasite's epidemiology). There are two main results. First, if despite its cost, resistance is fixed in the population, increasing the cost of resistance decreases the intensity of transmission. However, this epidemiological effect is weak if resistance is effective enough to be considered relevant for control. Second, if the cost of resistance prevents its fixation, increasing it intensifies transmission. Thus, the epidemiological effect of the cost of resistance cannot compensate for the lower frequency of resistant mosquitoes in the population. Overall, our conclusion remains pessimistic: so that genetic manipulation can become a promising method of malaria control, we need techniques that enable almost all mosquitoes to be almost completely resistant to infection.     

A mathematical model for the dynamics of malaria in mosquitoes feeding on a heterogeneous host population

We describe and develop a difference equation model for the dynamics of malaria in a mosquito population feeding on, infecting and getting infected from a heterogeneous population of hosts. Using the force of infection from different classes of humans to mosquitoes as parameters, we evaluate a number of entomological parameters, indicating malaria transmission levels, which can be compared to field data. By assigning different types of vector control interventions to different classes of humans and by evaluating the corresponding levels of malaria transmission, we can compare the effectiveness of these interventions. We show a numerical example of the effects of increasing coverage of insecticide-treated bed nets in a human population where the predominant malaria vector is Anopheles gambiae.     

Inverse Medea as a novel gene drive system for local population replacement: a theoretical analysis

One strategy to control mosquito-borne diseases, such as malaria and dengue fever, on a regional scale is to use gene drive systems to spread disease-refractory genes into wild mosquito populations. The development of a synthetic Medea element that has been shown to drive population replacement in laboratory Drosophila populations has provided encouragement for this strategy but has also been greeted with caution over the concern that transgenes may spread into countries without their consent. Here, we propose a novel gene drive system, inverse Medea, which is strong enough to bring about local population replacement but is unable to establish itself beyond an isolated release site. The system consists of 2 genetic components--a zygotic toxin and maternal antidote--which render heterozygous offspring of wild-type mothers unviable. Through population genetic analysis, we show that inverse Medea will only spread when it represents a majority of the alleles in a population. The element is best located on an autosome and will spread to fixation provided any associated fitness costs are dominant and to very high frequency otherwise. We suggest molecular tools that could be used to build the inverse Medea system and discuss its utility for a confined release of transgenic mosquitoes.     

A mathematical model for the dynamics of malaria in mosquitoes feeding on a heterogeneous host population

We describe and develop a difference equation model for the dynamics of malaria in a mosquito population feeding on, infecting and getting infected from a heterogeneous population of hosts. Using the force of infection from different classes of humans to mosquitoes as parameters, we evaluate a number of entomological parameters, indicating malaria transmission levels, which can be compared to field data. By assigning different types of vector control interventions to different classes of humans and by evaluating the corresponding levels of malaria transmission, we can compare the effectiveness of these interventions. We show a numerical example of the effects of increasing coverage of insecticide-treated bed nets in a human population where the predominant malaria vector is Anopheles gambiae.     

Plasmodium-mosquito interactions,phage display libraries and transgenic mosquitoes impaired for malaria transmission

Malaria continues to kill millions of people every year and new strategies to combat this disease are urgently needed. Recent advances in the study of the mosquito vector and its interactions with the malaria parasite suggest that it may be possible to genetically manipulate the mosquito in order to reduce its vectorial capacity. Here we review the advances made to date in four areas: (1) the introduction of foreign genes into the mosquito germ line; (2) the characterization of tissue-specific promoters; (3) the identification of gene products that block development of the parasite in the mosquito; and (4) the generation of transgenic mosquitoes impaired for malaria transmission. While initial results show great promise, the problem of how to spread the blocking genes through wild mosquito populations remains to be solved.     

Modelling Allee effects in a transgenic mosquito population during range expansion

ABSTRACT

Mosquitoes are vectors for many diseases that cause significant mortality and morbidity. As mosquito populations expand their range, they may undergo mate-finding Allee effects such that their ability to successfully reproduce becomes difficult at low population density. With new technology, creating target specific gene modification may be a viable method for mosquito population control. We develop a mathematical model to investigate the effects of releasing transgenic mosquitoes into newly established, low-density mosquito populations. Our model consists of two life stages (aquatic and adults), which are divided into three genetically distinct groups: heterogeneous and homogeneous transgenic that cause female infertility and a homogeneous wild type. We perform analytical and numerical analyses on the equilibria to determine the level of saturation needed to eliminate mosquitoes in a given area. This model demonstrates the potential for a gene drive system to reduce the spread of invading mosquito populations.     

Stocking of captive-bred fish can cause long-term population decline and gene pool replacement: predictions from a population dynamics model incorporating density-dependent mortality

Releasing captive-bred fish into natural environments (stocking) is common in fisheries worldwide. Although stocking is believed to have a positive effect on fish abundance over the short term, little is known about the long-term consequences of recurrent stocking and its influence on natural populations. In fact, there are growing concerns that genetically maladapted captive-bred fish can eventually reduce the abundance of natural population. In this study, we develop a simple model to quantitatively investigate the condition under which recurrent stocking has long-term effects on the natural population. Using a population dynamics model that takes into account a density-dependent recruitment, a gene responsible for the fitness difference between wild and captive-bred fish, and hybridization between them, we show that there is little or no contribution of recurrent stocking to the stock enhancement without a replacement of the wild gene pool by the captive-bred gene pool. The model further predicted that stocking of an intermediate level causes a reduction, rather than enhancement, of population size over the long term. The population decline due to stocking was attributed to the fitness disadvantage of captive-bred fish and strong overcompensation at recruitment stage. These results suggest that it would be difficult to simultaneously attain population size recovery and conservation of the local gene pool when captive-bred fish have fitness disadvantage in the wild, although caution is needed when applying the predictions from the simplified model to a specific species or population.     

Modelling Malaria Control by Introduction of Larvivorous Fish

Malaria creates serious health and economic problems which call for integrated management strategies to disrupt interactions among mosquitoes, the parasite and humans. In order to reduce the intensity of malaria transmission, malaria vector control may be implemented to protect individuals against infective mosquito bites. As a sustainable larval control method, the use of larvivorous fish is promoted in some circumstances. To evaluate the potential impacts of this biological control measure on malaria transmission, we propose and investigate a mathematical model describing the linked dynamics between the host–vector interaction and the predator–prey interaction. The model, which consists of five ordinary differential equations, is rigorously analysed via theories and methods of dynamical systems. We derive four biologically plausible and insightful quantities (reproduction numbers) that completely determine the community composition. Our results suggest that the introduction of larvivorous fish can, in principle, have important consequences for malaria dynamics, but also indicate that this would require strong predators on larval mosquitoes. Integrated strategies of malaria control are analysed to demonstrate the biological application of our developed theory.     

Nonconsensus West Nile virus genomes arising during mosquito infection suppress pathogenesis and modulate virus fitness in vivo

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV.     

Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood

Background

Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development.

Results

We have created two transgenic lines of Anopheles stephensi , a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A₂ (mPLA₂) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA₂ significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium -infected blood.

Conclusions

Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.     

Genetic engineering of malaria parasite resistance in vector mosquitoes

Malaria is the most significant vector‐borne disease and mostly affects people living in the lesser developed countries of tropical and sub‐tropical regions. Climate changes, rapid global transportation, immigration and invasion of exotic mosquito vectors bring the threat of introduction of the disease to developed nations. Sustainability of malaria control requires the discovery of therapeutic and prophylactic drugs, development of effective vaccines and control of vector mosquitoes. Drug development and vaccine research have been pursued aggressively over the past 20 years, and progress in novel approaches to vector control is now evident. Our long‐term objective is the production and utilization of strains of vector mosquitoes that are genetically refractory to the transmission of malaria parasites. These insects will be used to test the hypothesis that an increase in the frequency of a gene or allele that confers decreased vector competence to a population of mosquitoes will result in a reduction in the incidence and prevalence of malaria. Completed studies make it possible to develop strains of Anopheles mosquitoes expressing specific effector molecules that interfere completely with the transmission of the most lethal human malaria parasite, Plasmodium falciparum. Data are reviewed here that support the use of single‐chain monoclonal antibodies (scFv) that disable parasites in the midgut and hemolymph of transgenic mosquitoes.     

Optimal control strategy of malaria vector using genetically modified mosquitoes

The development of transgenic mosquitoes that are resistant to diseases may provide a new and effective weapon of diseases control. Such an approach relies on transgenic mosquitoes being able to survive and compete with wild-type populations. These transgenic mosquitoes carry a specific code that inhibits the plasmodium evolution in its organism. It is said that this characteristic is hereditary and consequently the disease fades away after some time. Once transgenic mosquitoes are released, interactions between the two populations and inter-specific mating between the two types of mosquitoes take place. We present a mathematical model that considers the generation overlapping and variable environment factors. Based on this continuous model, the malaria vector control is formulated and solved as an optimal control problem, indicating how genetically modified mosquitoes should be introduced in the environment. Numerical simulations show the effectiveness of the proposed control.     

Observance of period-doubling bifurcation and chaos in an autonomous ODE model for malaria with vector demography

We illustrate that an autonomous ordinary differential equation model for malaria transmission can exhibit period-doubling bifurcations leading to chaos when ecological aspects of malaria transmission are incorporated into the model. In particular, when demography, feeding, and reproductive patterns of the mosquitoes that transmit the malaria-causing parasite are explicitly accounted for, the resulting model exhibits subcritical bifurcations, period-doubling bifurcations, and chaos. Vectorial and disease reproduction numbers that regulate the size of the vector population at equilibrium and the endemicity of the malaria disease, respectively, are identified and used to simulate the model to show the different bifurcations and chaotic dynamics. A subcritical bifurcation is observed when the disease reproduction number is less than unity. This highlights the fact that malaria control efforts need to be long lasting and sustained to drive the infectious populations to levels below the associated saddle-node bifurcation point at which control is feasible. As the disease reproduction number increases beyond unity, period-doubling cascades that develop into chaos closely followed by period-halving sequences are observed. The appearance of chaos suggests that characterization of the physiological status of disease vectors can provide a pathway toward understanding the complex phenomena that are known to characterize the dynamics of malaria and other indirectly transmitted infections of humans. To the best of our knowledge, there is no known unforced continuous time deterministic host-vector transmission malaria model that has been shown to exhibit chaotic dynamics. Our results suggest that malaria data may need to be critically examined for complex dynamics.     

Spatial simulation of malaria transmission and its control by malaria transmission blocking vaccination

A simple, visual representation of spatial aspects of malaria transmission in successive snap-shots in time, is presented. The spatial components of the simulation involve (i) the identification of mosquito vector breeding sites of defined shape and area, (ii) the identification of a zone of malaria transmission determined by the shapes and areas of the vector breeding sites and the distance from these sites that the mosquitoes disperse, (iii) a human population dispersed in relation to the malaria transmission zone, (iv) perimeters around each individual human within which his or her infection can be transmitted by the local vector mosquitoes. The intensity of transmission within a malaria transmission zone is given by a number which is the number of new cases of malaria that each existing case will distribute through the human population within the duration of an infection. The simulation has been used here to examine the effects of vaccination against malaria transmission. Different levels of vaccine coverage are represented under endemic and epidemic malaria. The consequences of full or partial coverage of a zone of malaria transmission are also examined. The results are numerically compatible with the predictions of previous simple mathematical simulations of malaria transmission and interventions. The present simulation allows the nature of malaria transmission and the effects of interventions to be communicated easily and directly to an audience. It could have practical value in discussions of malaria control strategies with health planners.     

Day-to-Day Population Movement and the Management of Dengue Epidemics

Dengue is a growing public health problem in tropical and subtropical cities. It is transmitted by mosquitoes, and the main strategy for epidemic prevention and control is insecticide fumigation. Effective management is, however, proving elusive. People’s day-to-day movement about the city is believed to be an important factor in the epidemiological dynamics. We use a simple model to examine the fundamental roles of broad demographic and spatial structures in epidemic initiation, growth and control. We show that the key factors are local dilution, characterised by the vector–host ratio, and spatial connectivity, characterised by the extent of habitually variable movement patterns. Epidemic risk in the population is driven by the demographic groups that frequent the areas with the highest vector–host ratio, even if they only spend some of their time there. Synchronisation of epidemic trajectories in different demographic groups is governed by the vector–host ratios to which they are exposed and the strength of connectivity. Strategies for epidemic prevention and management may be made more effective if they take into account the fluctuating landscape of transmission intensity associated with spatial heterogeneity in the vector–host ratio and people’s day-to-day movement patterns.