Vector-Parasite Interactions and Malaria Transmission

Abstract

Malaria remains one of the world’s most devastating vector-borne diseases. During the complex sexual development of the malaria parasite in the mosquito, it is faced with physical and physiological barriers which it must surmount before it can be transmitted to a human host. Proof-of-concept studies using RNAi have unearthed several parasite molecules which are important for countering the immunity of its vector. Understanding the counter-adaptations between the parasite and its vector could inform novel public health intervention strategies. For instance, it could guide the transgenic construction of resistant mosquitoes in which mosquito factors that restrict the parasite growth have been enhanced and/or factors promoting parasite growth deleted so as to make them refractory to malaria parasite infection. Such strategies, when deemed feasible, could be combined with conventional vector control methods as well as treatment of infection with effective malaria therapy, to actualize the malaria eradication goal.

1. Introduction

Malaria is one of the world’s deadliest parasitic diseases affecting hundreds of millions of people worldwide. In 2020, the World Health Organization (WHO) reported an estimate of 241 million cases as compared to 227 million cases in 2019, with the number of deaths standing at 627,000 [1]. Due to the spread of insecticide-resistant mosquitoes as well as the development of Plasmodium falciparum resistant strains, control strategies are no longer as effective as they should. Thus, novel innovative strategies are required to combat this disease. A better understanding of the interactions between the mosquito and the malaria parasite may inform the development of new tools to control the disease. Transmission intervention by way of vaccines or transgenic mosquitoes may offer additional control strategies. Their development will, however, require the identification of valid molecular targets. The effective transmission of malaria requires specific compatibility between vector and parasite genotypes. Even within the susceptible Anopheles gambiae species (the most effective vector of the human malaria parasite), while some are resistant to infection, others, though unable to eliminate the infection, are capable of drastically reducing pathogen numbers [2, 3]. The mosquito molecules which interact with the malaria parasite to cause refractoriness in resistant strains have the potential to serve as targets for the development of novel transmission-blocking intervention strategies [4].

2. Malaria parasite life cycle stages

The female anopheles mosquito requires blood to nourish her eggs. As she sucks her victim’s blood, she secretes saliva and, if infected, injects sporozoites into the subcutaneous layer of the skin of her victim. The sporozoite travel to the liver where it invades hepatocytes. Here, it replicates asexually (mitotically) producing thousands of merozoites over a period of 6–15 days without causing any symptoms. Thereafter, the merozoites are released from the hepatocytes in the form of vesicles (merozomes). The vesicles disintegrate, releasing merozoites into the bloodstream to begin the erythrocytic stage of the disease. Within RBCs, parasites develop through ring, trophozoite, and schizont stages producing approximately 16 daughter merozoite per schizont. The schizont then ruptures in near synchrony with each other (unlike other human malaria parasites, P. falciparum does not exhibit distinct paroxysms) releasing hemozoin (malaria pigment) into the bloodstream of the victim which is responsible for the intermittent fever that accompanies the disease. The released merozoites invade new cells to initiate a new erythrocytic cycle. This cycle can go on and on resulting in host death from anemia unless the individual gets treated by an effective antimalarial therapy or the parasite gets killed by the immune system of the host. With each replication, some merozoites, instead of producing daughter merozoites, develop into male (microgametocyte) and female (macrogametocyte) gametocytes. Once gametocytes are picked up by a mosquito, transmission is initiated. The increased pH, lowered temperature as well as the presence of xanthurenic acid in the mosquito stomach, trigger the formation of the male and female gametes which fuse to form zygotes thereby initiating the sexual cycle [5]. The fusion of the gametes results in the formation of actively moving ookinete that migrates through the mosquito midgut to form oocytes containing thousands of sporozoites. The oocysts eventually burst to release these sporozoites which travel to the salivary gland of the mosquito for onward transmission.

3. Vector-malaria parasite interactions

3.1 Mosquito immune defenses

While the male anopheles mosquito feeds exclusively on plant nectar, in addition to feeding on plant nectar, the female anopheles mosquito requires blood to nourish and develop her eggs. During blood feeding, she’s exposed to malaria parasites (gametocytes), which must complete its complex developmental life cycle inside a mosquito host. The mosquito vector risks infection when there is physical injury to its cuticle or following cuticular degradation by the parasite. However, infection can be limited or reduced by mounting immune (innate and humoral) responses mediated by pattern recognition receptors and factors that trigger parasite killing via lysis, melanization (deposition of melanin on the surface of invading pathogens), and hemocyte-mediated phagocytosis. Further, many other mosquito molecules have also been reported to limit infection in the primary mosquito compartments which pathogens inhabit i.e. the midgut, the hemocoel, and the salivary glands.

3.1.1 Midgut

Upon ingestion of erythrocytes, cibarial armatures which mosquitoes use for RBC lysis are the first barriers faced by pathogens before they reach the midgut. Although the cibarial armature is effective in limiting infection by large metazoan parasites, it is not very effective at destroying protozoan parasites such as the malaria parasite [6, 7]. Transformation of ookinete to oocyst in the midgut is drastically reduced following lytic and melanization events. A number of molecules have been found to either facilitate or inhibit the parasite development within the midgut. They include the protein alanyl aminopeptidase N (AnAPN1); a surface recognition molecule which acts as a receptor for the malaria parasite in the mosquito midgut [8], a thioester-containing protein (TEP1), and leucine-rich repeat immune protein, (LRIM1) which recognizes the invading ookinetes at the basal lamina which surrounds the mosquito midgut and trigger immune responses [9, 10]. On the other hand, molecules such as C-type lectin 4 (CTL4), caspar, and cactus have been reported to negatively regulate the immune response of the mosquito, as silencing of these proteins resulted in decreased oocyst count [11, 12]. In A. gambiae midgut, (CTL4) and C-type lectin mannose-binding 2 (CTLMA2) negatively regulates the melanization of Plasmodium berghei ookinetes [13]. Further, the Serine protease inhibitor serpin 2 (SRPN2) also facilitates midgut invasion through inhibition of lysis and melanization [14, 15].

3.1.2 Hemocoel

The hemocoel is a nutrient-rich medium containing immune surveillance cells known as hemocytes. Hemocytes can be grouped into two sub-populations; granulocytes and oenocytoid. The granulocyte sub-population is capable of phagocytosing pathogens. Thioester-containing proteins (TEPs) are hemolymph proteins involved in the killing of Plasmodium ookinetes. The most studied (TEP) is the hemocyte-produced phagocytosis enhancer (TEP1). (TEP1) gets activated by complexing with the leucine-rich repeat containing proteins (LRIM1) and (APL1C) after which it opsonizes ookinetes for destruction by phagocytes [16]. Genetic variations in (TEP1) and (APL1C) are reported to affect mosquito immune competence against the parasite [3, 17].

Oenocytoids constitute the remaining population of the hemocytes. They are known to secrete enzymes of the melanization pathway (such as phenoloxidase and phenylalanine hydroxylase) used by mosquito to kill pathogens. Although the mechanism of pathogen killing by melanization remains unclear, it has been suggested that killing could either be the result of oxidative stress generated by unstable intermediates during melanogenesis or the result of starvation since melanization isolates the pathogen from the nutrient-rich hemocoel [18, 19]. In a literature search, Sreenivasamurthy et al. [20] identified a total of 22 molecules which play a role in melanization of ookinetes within the mosquito midgut.

3.1.3 Salivary gland

Sporozoites that successfully break through the mosquito immune defense system in the midgut lamina migrate to the salivary gland via the hemolymph. This they must do for transmission to occur. About 80–90% of sporozoites are reportedly lost during migration through the hemolymph. The mechanism by which this occurs is however not fully understood [21]. The invasion of the mosquito’s salivary gland has been reported to be triggered by effective and specific associations of sporozoite surface antigens such as thrombospondin-related anonymous protein (TRAP), with receptors such as saglin present on the salivary glands of the mosquito [14]. Using knockdown assays, Cui et al. [22] showed that four genes {AGAP006268 (peritrophin), AGAP002848 (Niemann-Pick Type C-2) (NPC-2), AGAP006972 (keratin-associated protein 16–1), and AGAP002851 (NPC-2)} play a crucial role in protecting the mosquito from parasite invasion whereas three other genes {AGAP008138 (uncharacterized), FREP1 (fibrinogen-related protein 1), and HPX15 (Heme peroxidase)} facilitated P. falciparum transmission to mosquitoes.

3.2 Parasite strategies for evading mosquito immune defenses

3.2.1 Midgut invasion

The malaria parasite must evolve mechanisms to evade the barriers put in place by the mosquito for successful completion of its life cycle which is an absolute requirement for parasite survival and effective transmission. A Plasmodium falciparum surface protein Pfs47 protects the parasite from the immune system of the mosquito in the midgut [23]. The result of the study by Molina-Cruz et al. [23] suggest that Plasmodium falciparum Pfs47 haplotypes dictate vector compatibility. The researchers demonstrated that A. gambiae fails to mount a proper immune response against several P.falciparum lines including NF54 and GB4 partly because of Pfs47 which mediates immune evasion by disrupting JNK/caspase-mediated apoptosis in the mosquito midgut [24]. Whereas, evasion of the complement-like response in Anopheles coluzzii, (a dominant species of the An. gambiae complex in West Africa) is mediated by the protein Plasmodium Infection of the Mosquito Midgut Screen 43 (PIMMS43) which is present on the surface of ookinete and sporozoite [25].

3.2.2 Salivary gland invasion

Once sporozoites are released from the oocyst, they migrate to the salivary gland via the hemocoel [21]. Salivary gland invasion is a key step in the life cycle of the parasite since changes that take place on the sporozoites surface proteins in the salivary gland enable them to invade the salivary gland of the mosquito and also to be successfully transmitted. The proteins, Plasmodium responsive salivary 1 (PRS1), epithelial serine protease (ESP), peptide-O-xylosyltransferase 1 (OXT1), and a serine protease inhibitor (SRPN6) have been shown to play crucial roles in parasite invasion of both midgut and salivary glands. While (SRPN6) limits salivary gland invasion by Plasmodium sporozoites [26], knocking down PRS1, ESP, retinoid and fatty-acid binding glycoprotein (RFABG) and (OXT1) have been reported to decrease oocyst and sporozoite numbers [27, 28, 29, 30]. Further, malaria parasites carrying mutations in conserved region II of the circumsporozoite protein (CSP) are unable to escape the oocyst [31]. Deletion of TRAP and LIMP (a highly conserved protein in Plasmodium parasites) severely impairs gliding motility which is important for salivary gland invasion [32, 33]. Whereas, although deletion of rhoptry neck protein 2 (RON) does not affect parasite’s gliding motility, salivary gland invasion is abolished [34].

4. Past, present, and future of transmission-blocking intervention strategies

Over the last two decades, the use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have been major contributors to gains in malaria eradication efforts [35, 36, 37, 38]. Even with the routine use of these key malaria interventions as well as effective malaria treatment with artemisinin-based combination therapies (ACTs), malaria-related mortality and morbidity remain unacceptably high with about half a million people losing their lives to the disease annually. Further, the development of resistance by the vector to insecticides as well as generation of parasite resistance to antimalarial drugs highlights the need for sustaining current gains and developing additional innovative control measures. Novel transmission-blocking intervention by vaccines or genetically engineered mosquitoes may provide a promising approach.

In 2013, the Malaria Vaccine Initiative (MVI) was rolled out. One of its key goals is the development of vaccines capable of interrupting transmission. Transmission- blocking vaccines (TBVs) are the result of efforts put in place by researchers to understand the interaction between the parasite and mosquito. Some TBVs are currently undergoing trials for efficacy and other key measures of success (Table 1). Alanyl aminopeptidase N (AnAPN1) is the leading midgut TBV immunogen [8]. (AnAPN1) is highly immunogenic and conserved between different anophelines. This makes it very attractive for vaccine development as vaccines prepared with this antigen should be active against all human malaria vectors hence saving the resources needed to develop specific targets for different Anopheles/Plasmodium species combinations [54]. Other midgut candidate molecules include carboxypeptidase [40], calreticulin [41], Croquemort SCRBQ2 [42] and myosin [43]. Candidate parasite molecules include those found on the surface of gametocytes and gametes (Pfs 2400, Pfs230, Pfs48/45) as well as zygotes and ookinetes (Pfs25, Pfs28) [55]. Vaccine against Pvs25 and Pfs25, which blocks P. vivax and P.falciparum respectively, are currently the leading molecule for a TBV [45, 56].

Vaccine candidate	Target	Stage of development/ClinicalTrials.gov identifier number	Reference
AnANP1	Midgut	Preclinical	[8]
FREP1	Midgut	Preclinical	[39]
Carboxypeptidase	Midgut	Preclinical	[40]
Calreticulin	Midgut	Preclinical	[41]
Croquemort SCRBQ2	Midgut	Preclinical	[42]
Myosin	Midgut	Preclinical	[43]
PfHAP2	Zygote	Preclinical	[44]
Pfs25	Midgut	Phase I (NCT01867463; NCT00295581)	[45]
Pfs28	Ookinete	Preclinical	[46, 47]
Pfs47	Midgut	Preclinical	[48]
Pfs48/45	Gametocytes & gametes	Preclinical	[49, 50]
Pfs230	Gametocytes & gametes	Phase I (NCT02942277)	[51]
Pfs2400	Gametocytes & gametes	Preclinical	[52]
Pvs25	Gametocytes, gametes, zygote & ookinete	Phase I (NCT00295581)	[53]
Pvs48/45	Gametocytes & gametes	Preclinical	[48]

Table 1.

Transmission-blocking vaccine candidates.

More recently, scientists have shifted attention from regular vector-control strategies (LLINs and IRS) to engaging advanced molecular tools such as CRISPR-cas9 to re-program the vector genome so as to make them refractory to the parasite (Figure 1). Gene drives skew the pattern of inheritance of genes creating mosquitoes that will either reduce mosquito populations or make mosquitoes less likely to spread the malaria parasite. Using such gene-drive systems in the laboratory, researchers have been able to transfer antimalarial effector genes to mosquitoes [57, 58]. Intriguing using similar allelic drive system in a Drosophila melanogasta model, Kaduskar et al. [59] were successful at reversing the most widely spread insecticide resistance mutation in anophelines (L1014F) (a mutation in the voltage-gated sodium channels of mosquitoes which make them resistant to pyrethroids) to susceptible wild-type genes (1014 L). The report of these researchers opens new vistas for vector control as it demonstrated that not only can insecticide resistance (IR) be reversed but that there is a relative negative fitness cost for the (L1014F) insecticide-conferring mutation as compared to the wild-type allelic variant. Thus, offering the opportunity to synergize the use of a gene drive that confers a bias inheritance of the preferred wild-type allelic variant with regular vector control methods. They went on to suggest that the identification of target site variants that would make the vector hypersensitive to insecticides will hold an even better promise. Further, they suggested the possibility of combining such gene drives for reversing insecticide resistance with other systems promoting refractoriness in mosquitoes [59].

Figure 1.

Breaking the cycle of malaria transmission.

Mosquito picks up gametocytes from the blood of an infected person. The sexual cycle is initiated due to the presence of xanthurenic acid (XA) as well as the low temperature and high pH of the mosquito stomach. The parasite then progresses through different stages and eventually forms sporozoites which are infective to humans. As depicted in the image above, parasite development can be interrupted at any stage of its sexual cycle either by using TBVs (candidate molecules are in boxes bordered by red broken lines) or genetic-based tools could be used to alter the expression levels of proteins crucial for mosquito infection (see boxes with broken black lines). Such tools capable of breaking the transmission chain could be incorporated as part of an integrated antimalarial strategy to eradicate the disease.

Although successful transgenic manipulation of mosquitoes has been achieved in the laboratories, their relative negative fitness in relation to wild-type populations is an important limitation for their relevance for large-scale use. For example, in a mark-release-recapture study in Burkina Faso recently, hemizygous genetically-modified (GM) sterile and non-transgenic sibling males of Anopheles coluzzii were released into a field in a controlled study. Recovered carriers of the GM trait had lower survival and were less mobile than their wild-type siblings [60]. Another shortcoming to the use of genetic-based vector control tools is that employing methods such as transposon-mediated transformation which modify only one allele of the desired gene, would spread the desired trait only to half of the offspring, and would eventually get eliminated in the wild population. This can however be overcome by employing gene drive systems such as CRISPR nuclease Cas9 which are capable of copying themselves to both gene alleles that will be inherited by all offspring, and thus spread more efficiently through a wild population [59]. Despite their great promise, scientists are wary of gene drive because they could cause irreparable damage since they permanently alter an entire population (Table 2). Further, issues bothering on their safety, governance, affordability, and cost-effectiveness need to be addressed (Table 2).

Strategy	Pros	Cons
TBVs	It can reduce child mortality in areas of malaria endemicity	Extremely long duration for vaccine preparation
	It can slow down the spread of mutant parasites thereby prolonging the efficacy of antimalarial drugs.	Lack of industrial partners which has hampered the progress of development [61].
	It can be combined with other multi-stage vaccines and contribute immensely to actualizing the eradication goal	Due to their mechanism of action, acceptability may be an issue. Although a recent survey in Bo, Sierra Leone reported that 96% of adults in that region will be willing to take TBVs [62], it remains to be seen if this will be the case when TBVs are eventually ready for use.
Gene drive	It can reduce or eliminate malaria by interrupting the transmission chain.	Safety concerns. The impact of gene drives on ecological habitat and the world at large is unknown. There is a possibility of off-targets that could cause serious harm to other organisms and even humans.
	Reduced use of insecticides thereby saving endangered species such as butterflies.	Ethical concerns of elimination of an entire population [63].
	Mosquitoes can be controlled in a more effective manner other than the conventional use of insecticides to which mosquitoes often become resistant.	Genetically modified mosquitoes could spread across borders creating more legal issues [64].
	It may reduce the economic and human cost of managing malaria.	Affordability. Many malaria endemic countries may be unable to afford it.
	It may be more long-lasing than conventional vector control methods since gene-drives can continue to spread through multiple generations without the need to re-introduce them	Cost-effectiveness. Although gene drive was seen to be the most cost-effective tool for the elimination of malaria in the Democratic republic of Congo using a mathematical model [65], some school of thoughts believe investing more money on health systems would be more beneficial and less risky than developing a gene drive technology for malaria.

Table 2.

Pros and cons of transmission-blocking intervention strategies.

5. Conclusion and future perspectives

The prolonged and repeated use of insecticides of a limited chemical class is a major contributor for the acquisition of resistance mutations in insecticide-target genes in insects. On the part of the malaria parasite, it also constantly evades antimalarial drugs by generating resistance mutations. Hence, the need to identify additional control measures that TBVs and genetics-based vector control tools may offer. Broad-spectrum malaria transmission-blocking vaccine antigens such as FREP1 [66] and (AnAPN1) offer attractive targets for the development of TBVs. However, due to the lack of industrial partners, the development and production of TBVs have stalled [61].

To generate transgenic mosquitoes, mosquito proteins that cause complete refractoriness in the vector upon silencing may be the target of choice. An ideal target molecule would be one that does not impose a relative negative fitness cost on the insect and one which could be used in combination with others targeting different stages of the parasite life cycle [67]. However, a major challenge with genome editing techniques is devising means to safely drive effector genes into mosquito populations in the field without causing harm to other organisms, including humans. Once safety concerns are addressed, such tools could be integrated with traditional vector control strategies, in addition to effective malaria treatment and good sanitation practices for the actualization of the eradication goal.

Conflict of interests

Authors declare no conflict of interests exists.

Author’s contribution

NE conceptualized the write-up. VO contributed to writing. NE contributed to writing, review and editing. Both authors have read and agreed to the final version of the manuscript.