Mosquitoes that can carry plasmodium are of increasing interest in cancer treatment. This is because experimental results show that infection with some species of plasmodium can help stimulate the immune system to better fight cancers such as Hepatocellular carcinoma (HCC). HCC accounts for between 85 and 90% of primary liver cancers. It is the third most common causes of cancer mortality worldwide. Plasmodium yoelii 17XNL infection significantly suppresses Lewis lung cancer (LLC) cell growth. This occurs via induction of innate and adaptive antitumor responses. Additionally, plasmodium infection inhibits tumor angiogenesis (Yang et al., 2017; Chen et al., 2011). Thus, although seemingly not important, mosquitoes that carry plasmodium can be used to help find ways to treat cancer. Thus, it is risky to eliminate whole species especially globally. Understanding mosquitoes is therefore a potential source of treatment methods and drugs which can turn out into billion-dollar industries and save millions of lives.
 
Methods of controlling or eradicating mosquitoes and malaria
Out of 460 different Anopheles species, 30 to 40 are vectors for the Plasmodium parasite. Methods for the control and treatment of malaria have been relatively successful, as evidenced by the decreasing malarial deaths (Cartolovni, 2017). The methods include vector control through insecticide-treated mosquito bed nets and indoor residual spraying. These have helped decrease the presence of Anopheles mosquitoes (Eckhoff et al., 2016; Bhatt et al., 2015). However, there is still residual transmission from mosquito vectors that feed outdoors or early in the evening. Malaria is treatable with anti-malarial drugs, which helps prevent the spread of the parasite by decreasing the number of parasites in the blood. The problem with vector control and treatment of malaria is the emergence of insecticide and drug resistance. Both the Anopheles mosquitoes and the Plasmodium parasite are showing resistance to the most commonly used chemical and pharmaceutical options to fight malaria such as pyrethroid. Artemisinin combination therapies (ACTs) are key to the treatment of P. falciparum malaria throughout the malaria endemic world. The emergence and geographic spread of artemisinin resistant P. falciparum represent a serious threat to global malaria control and to aspirations to eliminate malaria.  Resistance is attributed to mutations of the PfKelch13 gene. The gene has multiple independent origins across the Greater Mekong sub-region, which has motivated a regional malaria elimination agenda (Mernad et al., 2016). There are multiple mechanisms of insecticide resistance including changes to insecticide target molecules that render the insecticide unable to bind, behavioral changes leading to the avoidance of insecticide contact, thickening of the insect’s cuticle to prevent the insecticide reaching its target and detoxification of the insecticide before it reaches its target (metabolic resistance) (Barnes et al., 2017) ). In the malaria vector Anfunestus, pyrethroid resistance is mainly conferred by metabolic resistance associated with a major quantitative trait locus (QTL) at which two duplicated cytochrome P450 monooxygenases (CYP6P9a and CYP6P9b) are the main resistance genes (Wang and Jacobs-Lorena, 2013). The resistance to insecticides means that Malariaeradication requires new tools in addition to those currently deployed.
 
When mosquitoes take in a blood meal, the blood contains excess salts, such as potassium chloride that needs to be excreted via the kidney (Mernad et al., 2016). A team of researchers from Vanderbilt University Medical Center and Ohio State University developed a new class of insecticides that target the mosquito kidney. About 26000 compounds were screened for their ability to inhibit a potassium channel, Kir1, involved in urine production. It was noted that a compound called VU041 rapidly blocked the Kir1 channel activity (Pike et al., 2017). It is specific to mosquitoes and does not affect any mammalian potassium channels tested. The team monitored mosquitoes to assess kidney function and observed that when untreated mosquitoes consumed a blood meal, their abdominal diameter immediately doubled, and then decreased over the next 24 hours. In contrast, the abdominal diameters of mosquitoes treated with VU041 increased but did not decrease, suggesting that the impairment of kidney function. The mosquitoes kept on increasing in weight until they burst. VU041was found to reduce egg laying after blood feeding suggesting that VU041 can be used to control mosquito populations.
 
A recently developed strategy is to use Metarhizium anisopliae, a fungus that naturally attacks mosquitoes, as mosquito-specific biopesticides (Lovetteet al., 2019). The mosquitoes must acquire the fungus soon after becoming infected with the malaria parasite. Rather than developing fungi that rapidly kill the mosquito, the fungus is genetically modified to block Plasmodium development inside the mosquito. Metarhizium pingshaense provides an effective, mosquito-specific delivery system for potent insect-selective toxins. After invading a mosquito, the transgenic fungi produce small molecules such as the human anti-malarial antibody and a scorpion antimicrobial toxin (Pike et al., 2017). When mosquitoes that are heavily infected with P. falciparum are sprayed with transgenic fungi, they have a significantly reduced parasite development. The transgenic fungus does not significantly affect mosquito survival when compared to the wild-type fungus. Hence the transgenic fungi do not lead to rapid mosquito resistance when used in the field.
 
Changing the mosquito’s ability to support the life cycle of Plasmodium parasites can be achieved through genetic engineering of the mosquito gut, making it too hard for Plasmodium to survive (Wang et al., 2013). One way of achieving that is is to genetically modify the Anopheles mosquito to make part of the mosquito’s gut, where the Plasmodium parasite normally grows, into an inhospitable habitat where Plasmodium cannot survive. This means that the mosquitoes will need to have new effector genes introduced so they can be expressed as anti-Plasmodium proteins within the mosquito’s gut, making the environment uninhabitable for the Plasmodium to survive. Introduction of the effector genes into the mosquito includes using fungi or viruses that already infect mosquitoes or using bacterial symbionts that already inhabit the mosquito gut (Carvalho et al., 2015). Another way is to modify the symbiotic gut bacteria. When the modified bacteria enter the mosquito gut, the mosquito begins to express the anti-Plasmodium proteins, making the mosquito gut inhospitable to Plasmodium parasites (Pike et al., 2017).
 
One of the recent technologies of genetic engineering to control malaria involves decreasing the mosquito population so there are fewer mosquitoes to transmit malaria. The genetic modification of the mosquito vector employs CRISPR technologies while the dissemination of the sterility gene to run in the populations uses the gene drive technology (Tuna et al., 2019). An example of a potential method has been successfully tested in Aedes mosquitoes to combat dengue. The approach involves genetically modifying male mosquitoes so their offspring never matured, dying before they were able to transmit dengue (Carvalho et al., 2015). The use of these transgenic sterile males resulted in a wild mosquito population decrease of 80-95%. The study showed that genetically engineering sterile mosquitoes can drastically decrease the mosquito population in an area.
 
Mosquitoes can be genetically modified to alter the expression of their anti-Plasmodium immune genes in a population with wild-type mosquitoes. Multiple GM Anopheles stephensi lines can be created that are resistant to Plasmodium falciparum due to the up-regulation of mosquito immune genes in the midgut or fat body after a blood meal, using the carboxypeptidase (Cp) or vitellogenin (Vg) promoter, respectively (Mernad et al., 2016). These strains will possess elevated anti-Plasmodium and antibacterial activities through either the immune-deficiency pathway–associated NF-κB transcription factor Rel2 or the Down syndrome cell-adhesion molecule (AgDscam) splice form AgDsPf (Swale et al., 2016). The GM lines can be backcrossed with the original wild-type stock for five generations and be continually reared under the same conditions to ensure the same genetic and environmental background. Genetically modified mosquitoes with increased immune activity in the midgut tissue will not have an observed fitness disadvantage and will show reduced microbial loads in both the midgut and reproductive organs. These changes result in a mating preference of genetically modified males for wild-type females, whereas wild-type males will prefer genetically modified females. These changes will foster the spread of genetic modification in a mosquito population and help control mosquito populations (Pike et al., 2017).
 
Potential of Gene drives in eradicating mosquitoes and malaria
Gene drives can be characterized by the rate of spread, species specificity, fitness cost, susceptibility to resistance, removability, and reversibility. Engineered gene drives can be divided into the modification drive types designed to spread genomic changes and or genetic payloads throughout a population, suppression drive types designed to reduce or eliminate the population of its target organism and reversal gene drive types which induce further changes that may undo a phenotypic alteration caused by the initial gene drive. The envisioned goal for applying gene drives is to reduce or eliminate vector mosquito populations or to render them less competent to transmit pathogens. With a gene drive, not only is it possible to alter an organism’s gene, but it is also possible to insert in the genome the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) copy-paste system which includes the gRNA and Cas protein.  This allows the gene alteration to self-replicate in subsequent generations. As an example, when an altered mosquito mates with a wild mosquito, the offspring receives an altered chromosome and a wild chromosome from each parent. The CRISPR system inherited from the altered parent will cut the wild gene inherited from the wild parent and copy the altered gene into the offspring’s genome along with the gene drive. The offspring then carries two copies of the altered gene, ensuring its transmission to the next generation. When a new generation of altered mosquito mate with the wild types, the process will repeat itself allowing the alteration and the gene drive to spread in the whole population. The gene drive, therefore, appears to be a reliable mechanism for propagating altered genes, which in theory would allow gene alterations to persist in nature and permanently change the target population and possibly an entire species. Computational modeling based on other gene drive systems suggests that the type of drive that can be achieved with the CRISPR/Cas9 system can be so effective that release of low numbers of modified mosquitoes into the environment could result in establishment of the genetic modification in the natural interbreeding population (Tuna et al., 2019; Eckoff et al., 2016).
 
One way of applying gene drives in mosquito control is via the use of the Wolbachia gene drive. Wolbachia is a naturally occurring bacterium that was previously found to block the development of Plasmodium parasites in mosquitoes. Wolbachia can be transmitted by an infected female insect to the offspring. Uninfected females that mate with infected males rarely produce viable eggs, a reproductive dead end that gives infected females a reproductive advantage and helps the bacteria spread quickly. Wolbachia were successfully used in a field trial to control dengue, another mosquito-borne disease (Swale et al., 2016). However, the bacteria do not pass consistently from mother to offspring in Anopheles mosquitoes, which spread malaria. The researchers injected a strain of Wolbachia derived from another type of mosquito into A. stephensi embryos. Once matured, the adult females mated with uninfected male mosquitoes to create a stable Wolbachia infection that persisted for 34 generations (the end of the study period). Uninfected females rarely produced viable eggs with infected males. The researchers found that Wolbachia infection reduced the number of malaria parasites in both the mosquito midgut and salivary glands. Wolbachia infection causes the formation of reactive oxygen species, which inhibit parasite development.