Finding Malaria’s Achilles Heel: Intervention Strategies Informed by Life History and Evolution

Summary

Malaria is a devastating disease caused by the Plasmodium parasite that has not responded to many interventions. Disease control methods that target only one facet of the disease, like antimalarial drugs and pesticides, leave opportunity for evolving resistance. Malaria’s long evolutionary history among humans and its mosquito vector has resulted in intense selection pressure on each organism to evolve traits that confer survival. This coevolution makes malaria a challenging disease to eradicate; therefore, achieving malarial control requires an extensive understanding of malaria’s evolutionary and life history. Malaria’s life and evolutionary history reveal that arresting mosquito longevity is the key to malarial control. This paper will demonstrate that an effective intervention strategy must take a multifaceted and ecologically conscious approach towards targeting the mosquito vector.

Introduction

Malaria is a vector-borne pathogen that has remained a prevalent health issue in many parts of the world. In 2018, there were 228 million clinical episodes reported, 405,000 of which resulted in fatality [1]. Sub-Saharan Africa bears the brunt of this mortality burden; in 2020, 95% of malaria cases occurred in Sub-Saharan Africa, 96% of which were fatal and 80% of which occurred in children under five years of age [2]. Unfortunately, the deadliest malaria parasite, Plasmodium falciparum, is the most prevalent malaria parasite in Africa [3]. P. falciparum is a difficult parasite to target due to its complex life cycle and long history of coevolution with its mosquito vector, Anopheles gambiae, and its human host, Homo sapiens [4]. In humans, evolutionary pressures of P. falciparum encouraged the evolution of the hemoglobin S variant, which protects against malaria in its heterozygous state and causes sickle cell anemia in its homozygous state. In the P. falciparum genome, many large portions are dedicated entirely to immune evasion and host-parasite interactions [5]. Analysis of A. gambiae’s genome suggests that selection pressure from P. falciparum fostered the creation of an incredibly efficient and highly adapted vector. Yet, this long history of coevolution between malaria and its hosts does not necessarily suggest that the relationship between them is beneficial for all; rather, it could be an example of the Red Queen hypothesis. This hypothesis states that both predator (malaria) and prey (mosquitoes and humans) must continuously adapt to each other’s advantageous mutations  to continue out-competing each other so that neither competitor goes extinct [6]. This continuous race creates limitations for drug and insecticide based interventions due to Plasmodium’s and Anopheles’ abilities to quickly evolve resistance. Thus, an efficacious malaria control model needs to mitigate this risk by targeting malaria from multiple fronts. This can be achieved by creating a multifaceted approach rooted in ecological and evolutionary knowledge of the malaria parasite and its hosts. 

Malaria Parasite Life History

Life Cycle Overview

The malaria parasite has a complex life cycle composed of intricate host interactions. When an Anopheles gambiae mosquito bites a human host, it ingests Plasmodium falciparum gametocytes with its blood meal [7]. Once inside the mosquito vector, the malaria gametocytes meiotically divide into gametes, which undergo sexual reproduction [8] to form a zygote. This fertilization event produces an elongated motile cell called an ookinete [9, 10]. The ookinete migrates from the midgut lumen through the midgut epithelial layer to the basal lamina, where it becomes sessile and transforms into an oocyst [10]. The oocyst is the only Plasmodium life cycle stage that grows extracellularly and forms sporozoites. Once mature, sporozoites burst out of the oocyst and into the hemocoel. Then, the sporozoites use the hemolymph circulation system to reach the basal lamina of the mosquito salivary glands, where they wait to be released into a mammalian host in its next blood meal (figure 1) [10].

Once a mosquito bites a human host, the sporozoite’s journey to the human liver begins. From the site of the bite, sporozoites migrate through the human dermis (skin) [11] and find the bloodstream on their own [10]. Once sporozoites reach the liver, they traverse Kupffer cells, which are liver macrophages that protect the liver against foreign bodies. Then, they invade liver cells called hepatocytes, where they replicate by up to 40,000-fold and differentiate, resulting in asexual blood-stage merozoites [4]. Merozoites enter the bloodstream via hepatocyte-derived vesicles, and begin a cycle of red blood cell invasion, replication, and rupture (figure 2). The invasion of red blood cells causes the host’s red blood cell count to dwindle, resulting in anemia, which causes many of the commonly recognized symptoms of malaria. Some merozoites break out of the red blood cell invasion cycle and commit to developing into gametocytes that are picked up by the next mosquito during a blood meal [12]. Once ingested by the mosquito again via blood meal, the gametocytes undergo sexual reproduction and the life cycle starts again.

Bottlenecks

The malaria parasite encounters many obstacles during its life cycle in mosquito hosts, resulting in the occurrence of multiple bottlenecks, which are stages during the Plasmodium life cycle with significantly lower survival rates. The first major bottleneck occurs during the gametocyte stage. Gametocytes ingested by the mosquito transform into gametes in the midgut and are vulnerable to phagocytes consumed with the blood meal [13]. Specific temperatures and pH changes initiate the transformation of gametocytes into macro and micro gametes. Without these conditions, sexual reproduction would not be possible.

If conditions are optimal for gamete production, the resulting zygote and the subsequent ookinete still have a long journey ahead with multiple bottlenecks before it becomes an infectious sporozoite. Ookinetes must breach the midgut epithelium in order to transform into oocysts [13]. This rupture in the midgut allows bacteria to escape, triggering an antibacterial immune response from the mosquito, which can kill ookinetes caught in the cross-fire [4]. The surviving ookinetes turn into oocysts, which depend on the mosquito for nutrients and laminin during their development [10]. Mature oocysts divide via endomitosis to produce sporozoites that must escape the oocyst with their limited, weak motility. These immature sporozoites use the mosquito hemolymph circulatory system to reach the salivary glands, a journey that only 25% of them will complete [13]. Despite the debilitating bottlenecks a malaria parasite faces during its transformation from gametocyte to sporozoite, malaria remains a global health issue. However, within this complex life cycle lies the clues that reveal how natural selection acts on P. falciparum, which will help inform the employment of effective intervention strategies.

Malaria’s Secrets to Success

The Plasmodium parasite compensates for bottlenecks in its life cycle by maintaining a large, genetically diverse population. There is intense selection pressure on the malaria parasite during transmission stages to increase the frequency of beneficial mutations [14]. To observe this selection pressure, the change in the frequency of a beneficial mutant allele throughout the malaria parasite’s life cycle can be modeled during two stages of the parasite’s life cycle when it experiences intense selection pressure to survive: during host red blood cell stages and during transmission. These following selection scenarios were modeled: selection only takes place in the host organism (host selection), selection is impacted during host to vector transmission (transmission selection), and selection takes place in the host combined with host to vector transmission (host/transmission selection). When modeling allele frequency changes over five generations under host selection, transmission selection, and host/transmission selection, alleles are fixed the quickest during host selection and host/transmission selection (figure 3). This indicates that if there is a selective advantage in the host stages within the initial generations, the frequency of beneficial mutations increases faster. Then, the transmission advantage becomes dominant, so the allele frequency in transmission and host/transmission selection models will increase more quickly.

When modeling allele frequency changes until fixation, beneficial alleles become fixed the most rapidly under transmission and host/transmission selection (figure 4). This demonstrates that transmission advantages have a greater influence on the course of beneficial mutations than selective advantage in the red blood cell stages [14]. A beneficial allele becomes fixed most quickly in the host/transmission model, implying that the beneficial mutation has a within-host selection advantage and a between-host transmission advantage. In the five generation model, an allele is fixed after a few generations in the mammalian host because of asexual reproduction [15]. However, selection inside the host is only important when mutations are still being partitioned [14], thus, transmission selection will have a more dominating effect on allele frequency (figure 4).

The immense pressure exerted by natural selection ensures the fixation of mutations that confer the highest transmission advantage, thereby overcoming the bottlenecks it encounters during its life cycle. With every blood meal, the mosquito ingests multiple groups of gametocytes that genetically recombine and create a diverse population upon which intense selection pressure is exerted. Therefore, this genetic recombination process becomes a melting pot for beneficial mutations. Protecting this portion of the life cycle optimizes malaria’s transmissibility, which makes it such a successful pathogen.

Evolutionary History of A. gambiae

Unraveling Anopheles gambiae’s evolutionary and life history can inform strategies for effective malaria control. Anopheles gambiae is the most efficient malaria vector that transmits malaria today [16]. The Anopheles genome and evolutionary history reveal traits that make A. gambiae the dominant malaria vector. By comparing Anopheles and Drosophila, two highly adapted members of the Diptera order [17], the traits that reveal information about Anopheles’ evolutionary history can be easily identified. Compared to the Drosophila genome, the Anopheles genome evolved more dynamically, exhibited a five times higher rate of gene gain and loss, and had a higher rate of rearrangement on the X chromosomes (one of the two chromosomes that determine sex) than the autosomes (all of the chromosomes excluding the sex chromosomes) [18].

A higher rate of rearrangement on X chromosomes than autosomes contributes to Anopheles’ ability to quickly introduce and increase the frequency of beneficial alleles in its lineage. This phenomenon is called faster X, and is caused by an increased rate of nonsynonymous substitutions on the X chromosome [19]. These nonsynonymous substitutions change the resulting protein [20]. If the X chromosome is mutated at a higher frequency than the autosomes, hemizygous males are more likely to express the mutation and pass it on to their offspring [19]. This occurs because males have one X and one Y chromosome, making them hemizygous for the genes on each chromosome. During sexual reproduction, the female will pass on only one of her two X chromosomes, and the male, being hemizygous, will only be able to pass on either his X or Y chromosome. So a mutation on the X chromosome will be easily passed on by the male. Assuming an equal mutation rate of beneficial alleles on X chromosomes and autosomes, the rate of adaptive evolution will be higher for the loci on the X chromosome because of the hemizygous males [21]. Comparing Drosophila and Anopheles reveals that Anopheles’ high rate of adaptive evolution contributes to its efficiency as a malaria vector.

Gene gains and losses at multiple points in the Anopheles evolution can suggest extreme selection pressure; this selection pressure likely stems from the presence of Plasmodium in Anopheles dense regions [18]. P. falciparum requires a long lived, anthropophilic vector [22] and exerted intense selection pressure on A. gambiae to facilitate the evolution of these traits [18]. Female A. gambiae mosquitoes are a perfect match due to their month-long life cycle [23] that allows for frequent feeding on human hosts [24]. The mosquito vector’s longevity enables P. falciparum gametes, stemming from a variety of human hosts, to sexually reproduce inside the mosquito midgut, thus facilitating gene flow. This increased gene flow facilitated by frequent host feeding and intense transmission selection balances out malaria’s bottlenecks and suggests that host longevity is the key to successful transmission.

Control Strategies

The Fibrinolytic System

The coevolution of humans and malaria gives insight into the weak points of malaria’s immune-evasion tactics, which reveals promising targets for intervention strategies. One potential intervention target is the human fibrinolytic system, which is responsible for removing fibrin, an insoluble protein and the main component of blood clots [25], from blood vessels to prevent clots [26]. Two important enzymes in the fibrinolytic system are plasminogen and plasmin [27]. Plasminogen is a protein that can be cleaved into plasmin by a urokinase plasminogen activator (uPA) or a tissue plasminogen activator (tPA) [28]. Plasmin is an enzyme that facilitates clot lysis [29], also called fibrinolysis, which is the degradation of blood clots [30]. Human plasminogen is commandeered by Plasmodium parasites to aid their migration through the multiple proteinaceous matrices inside the mosquito vector and mammalian host throughout their life cycle [31]. Without these enzymes, Plasmodium falciparum would not be able to sexually reproduce in the mosquito or traverse through the human dermis and liver. Given plasminogen’s importance to P. falciparum’s life cycle, targeting the fibrinolytic system could be an effective malaria intervention, but should be used in tandem with other intervention strategies to avoid the evolution of resistance within the malaria parasite.

Genetically Modified Mosquitoes

Genetically modifying mosquitoes is a propitious malaria control strategy that targets the mosquito vector. For example, a mosquito could be genetically modified to target the fibrinolytic system by inhibiting plasminogen activators via the secretion of human plasminogen activator inhibitor 1 (huPAI-1) [11]. Two modes of genetic modification are genetic engineering and gene drives. Genetically engineered (GE) and gene drive (GD) mosquitos both involve the introduction of a new trait into the wild population. A GE organism contains an engineered gene that can be introduced into the normal population via hybridization [32]. Then, natural selection can act on the GE organism the same as it would act on the non-GE organism. GD organisms, however, are based off of the concept of natural gene drives, which is a phenomenon where a genetic element is able to bypass mendelian inheritance and is inherited by more than half of an organism’s offspring (figure 5) [33]. There are many types of gene drives found in nature; for example, homing endonuclease genes are found in many microorganisms [34]. These genes encode endonucleases that are inserted into a specific site in a homologous chromosome to be cut at the site of insertion. Homology-directed repair pathways then copy the endonuclease genes into the breaks they created. This natural gene drive system laid the groundwork for developing engineered gene drive systems, in which DNA cleavage could be directed to desired sites using the CRISPR-Cas9 endonuclease and an engineered guide RNA. In the field of malaria control, an engineered gene drive system could be used to introduce specific genetic elements into mosquito populations that diminish their vectoral capacity.

The goal of GE mosquitoes is vector suppression, which is achieved by intraspecific gene flow between the modified and wild populations [36]. A lack of gene flow prevents the engineered gene from infiltrating the wild population. For example, gene flow could be inhibited by reproductive isolation or competitive exclusion. The mechanism for vector control with gene drives is slightly different. Synthetic gene drives can either modify or suppress a population (figure 6) [35]. Population suppression involves the addition of a gene that compromises the survival or reproduction of the target population, while population replacement involves the introduction of a gene that decreases the survival of the wild population and enables the modified population to replace it.

Though genetically modified mosquitoes are a promising intervention, their potential to cause environmental and ecological harm must be carefully considered prior to implementation To mitigate ecological damages from GD mosquitos, they can be designed to be self-limiting, meaning that there is some biological or molecular confinement that limits the geographic or temporal range in which they remain effective [35]. To understand the consequences of and opportunities to control genetically engineered mosquitoes, a model can be employed that demonstrates hypothetical effects of GE mosquito release. Ecological effects from GE mosquitoes can occur in two phases: the transitory phase and the steady state phase [36]. These phases can be analyzed in the context of eight different pest control scenarios (figure 7).

Curves I-III are representative of current genetically engineered insect management strategies (figure 7). For example, curve I could represent the release of sterile genetically modified mosquitoes, which would cause an initial increase in population density, followed by a decline and eventual plateau because the modified mosquitoes do not reproduce and thus stagnate the population. Curve II shows a population that increases slightly above the initial density, then returns to and plateaus at the initial density. This could describe a genetic engineering strategy that involves the release of genetically engineered individuals that replace the wild population without changing the wild population's initial population density. Curve III shows a population that increases slightly, then rapidly decreases. This curve can represent a population suppression strategy that involves the release of a genetically engineered population that infiltrates and decreases the wild population. Modeling and identifying the changes in population density in the transitory phase helps identify the adverse evolutionary or ecological effects in the steady state phase. For example, changes in population density during the transitory phase can cause changes in gene flow; a decrease in population density results in reduced gene flow and makes the population more vulnerable to genetic drift. This evolutionary effect is important for GE organisms because a smaller population will be affected more strongly by genetic drift, which could lead to the loss of the GE gene. 

A successful genetically modified mosquito intervention needs to account for possible ecological and evolutionary consequences. For example, the genetically modified mosquito population could out-compete the natural population, but then become invasive and increase in population size. Eliminating consequences like this can be done by either creating a self-limiting gene drive mosquito or installing ecological parameters that control the negative impacts of the introduction of genetically engineered mosquitoes. These genetic interventions should also be used in accordance with other interventions that target malaria from different fronts to mitigate ecological consequences and prevent the possibility of evolutionary resistance. 

Larvivorous Fish: A lesson from the Brazil Campaign

Vector control is one of the most challenging aspects of malaria control due to the constant evolution of intervention-evasion behaviors. However, some regions have been able to achieve complete vector control, and studying their methods could inspire efficacious malaria control strategies in Africa. For example, the campaign on the northeast coast of Brazil is one of the only campaigns that has ever achieved complete eradication of a major African malaria vector [37]. The northeast coast of Brazil has ecological conditions that are similar to the regions of Africa that experience a high prevalence of malaria. These ecological conditions, including temperature, humidity, precipitation, and mosquito larva habitat features, make the Brazil campaign a useful case study that can inform the employment of effective vector control strategies to suppress malaria in Africa. Vector eradication was made possible by the use of an effective larvicide, a large workforce, consistent monitoring of the mosquito’s ecological niche, and adaptation according to vector ecology. However, the use of larvicide should not be repeated in Africa. In Brazil, larvicide was effective for mosquito eradication, but it was a toxic chemical that caused many cases of human poisoning [37]. The key takeaway from the use of larvicide in the Brazil campaign is that focused larval control made a significant difference in the effectiveness of their eradication program. P. falciparum needs a stable population of healthy, adult female mosquitoes that live long enough to support the ten day development period required for sporozoites to become infectious. Therefore, killing mosquitoes in their larval stage will directly impact A. gambiae’s vectoral capacity by reducing host longevity.

A different way to directly interfere with mosquito longevity without larvicide is to introduce predators. A. gambiae mosquitoes are preyed upon in every stage of their lives [16]. The presence of a predator alone creates physiological stress that negatively impacts mating, fecundity, body size and survival [38]. Thus, the introduction of predators could directly contribute to the suppression of the mosquito vector. Larvivorous fish are an advantageous choice for achieving vector suppression of A. gambiae via predation. Adult A. gambiae mosquitoes are ephemeral and disaggregated, making them a challenging target for predation-based interventions [16]. Their preferred breeding habitats are small, shallow, sunlit and often temporary pools that are usually created by poor development or agricultural practices. However, mosquitoes also persist in deeper and more permanent bodies of water. If the shallow, temporary pools are managed and cleared by improved development practices, then the larger, permanent habitats could be the target for a predation-based intervention.

The introduction of larvivorous fish into A. gambiae larval habitats can be a sustainable and ecologically conscious method for reducing mosquito longevity. The use of larvivorous fish to target the larval stage of A. gambiae eliminates many of the risks associated with targeting the adult stage; for example, it does not involve a drug or insecticide that the mosquitoes can evolve resistance towards, and it leaves little opportunity for behavioral adaptation. Fish are already the most widely utilized biological control agent of mosquitoes, and their implementation in sub-Saharan Africa could be an integral step towards complete malaria control [39]. Due to their potential to cause ecological harm, many studies have been conducted to evaluate native fish species on their larvivorous potential and risk of ecological damage. Native fish species are preferable to nonnative species because they are less likely to become invasive. A study done in the Republic of Benin evaluated the use of larvivorous fish to suppress the population of A. gambiae larvae under laboratory conditions. This study used the indigenous larvivorous fish species, Hemichromis fasciatus, and measured the fish species’ larva-eating capacity. The study found that H. fasciatus had a high larva-eating capacity in both fed and unfed conditions; though they reduced a higher proportion of the larval population when they were not given fish food beforehand, they still reduced a significant proportion of the larval population after already being fed. The results of this study highlight the importance of selecting a fish species that is already native to the area of interest and will actively eat larvae even in the presence of other food sources. 

If chosen wisely and used in tandem with other vector control strategies, the use of larvivorous fish could make a significant improvement in current malaria control tactics. Additional control strategies are also advisable to preserve the efficacy of the larval control method. Targeting the vector species on multiple fronts will prevent natural selection from exerting intense pressure on the vector to quickly adapt a trait that enables them to evade a specific intervention mechanism. In addition to the characteristics identified in the Benin study, a larvivorous fish species suitable for this intervention also needs to be a small, hardy, drought-tolerant species that breeds prolifically [40]. These parameters will reduce the potential for ecological and evolutionary risks associated with the introduction of a predator into an ecosystem by minimizing the risks of the species outcompeting other aquatic species, dying out in the presence of poor environmental conditions, and having little impact on vector population suppression because of low fecundity. 

Models are useful for visualizing the relationship between mosquito lifespan and human biting rate, which can help inform strategies for vector management. For example, to measure the efficacy of larvivorous fish as a vector management strategy, we want to reduce R₀, the basic reproduction ratio. R₀ represents the number of infected individuals that arise from the introduction of a single infected individual into a susceptible population [41]. R₀ measures the success of a pathogen, and for vector borne diseases like malaria, it depends on the following factors, derived from the Ross-Macdonald model: the ratio of mosquitoes to humans, the mosquito biting rate, the pathogen transmission efficiencies, the daily survival rate of mosquitoes, the recovery rate in humans, and the duration of the extrinsic incubation period [42]. An R₀ value of less than one indicates that an infected host is infecting less than one susceptible host, so the disease has a low probability of infecting enough hosts to remain prevalent in the population [41]. Changing R₀ requires changing the factors that it depends on, like those included in the Ross-Macdonald model. For example, the introduction of larvivorous fish would impact the ratio of mosquitoes to humans and the mosquito biting rate; if those factors are reduced, R₀ will also be reduced. An effective vector management strategy has the potential to push malaria to extirpation if it can reduce the mosquito biting rate of humans to under 0.16 (figure 8) [43]. This model demonstrates that the introduction of larvivorous fish used alongside another vector management strategy can reduce the human biting rate of mosquitoes, which will bring the reproduction rate to less than one, thus reducing the transmissibility of the pathogen and paving the way towards disease eradication.

Conclusion

The disproportionate prevalence of malaria in Africa calls for the implementation of effective solutions. This immense malaria burden in Africa is largely due to its geography, climate, agricultural practices, and poor health infrastructure; all of which exacerbate transmission rates [37]. In the past, reliance on DDT and pesticide treatments to control the mosquito vector resulted in evolved resistance and environmental damage. The extensive use of antimalarial drugs has also resulted in evolved resistance due to malaria’s complex life cycle and enmeshed host relationships [44]. Furthermore, resistance to one antimalarial drug often manifests resistance in similar drugs [45]. Other common and less damaging interventions include bed nets and indoor pesticide spraying, but the use of these techniques in isolation of other control methods and improvements to infrastructure will not result in complete malaria control. A thorough investigation of weaknesses in malaria’s life history reveals that the disease relies on the A. gambiae vector to facilitate efficient transmission and on host biomaterials to facilitate movement through host barriers. These weaknesses are prime targets for multiple interventions that, when used together, can create a successful and multifaceted approach to control malaria transmission.

About the Author: Emily Jajeh

Emily Jajeh is a Global Disease Biology major with an emphasis in disease ecology. She chose this major because of her lifelong interest in disease, which has evolved into a passion for One Health and disease ecology. In her third year at UCD, she spent three months in Bwindi, Uganda, volunteering with Bwindi Community Hospital and working with the head of the Community Health department to complete her practicum project about malaria. She wrote this paper in preparation for her trip so that she could learn more about malaria from a disease ecology perspective and begin her project with a well-rounded understanding of the disease. For her practicum project, she conducted a case study in the Kanyahsande village to reveal potential risk factors for malaria in children under five years old, and assess how well parents were able to identify malaria symptoms in their children under five years and seek proper treatment. Her research in Uganda was largely influenced by this paper because its disease ecology focus helped her create a thorough case study that laid the foundation for many multifaceted approaches for future outreach and research.

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