Symbionts and gene drive: two strategies to combat vector-borne disease

Safe and sustainable approaches for mosquito control are critical due to the global increasing burden of mosquito-transmitted diseases.Novel control approaches based on symbionts are currently proposed to modify or suppress mosquito populations and Wolbachia-based methods have already achieved some success in field trials.Transgenic mosquitoes carrying gene drives that spread through populations are a promising control approach to block disease transmission or suppress vector species.Transgenic-based approaches potentially offer more power and flexibility, but symbiont-based approaches are usually more socially accepted and well-developed. Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineering-based approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies. Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineering-based approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies. Mosquitoes can be found almost anywhere in the world, but in the tropics and subtropics, half of the world's population is under the threat of mosquito-borne pathogens such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever, West Nile virus (WNV), malaria, and filarial nematodes [1.World Health Organization Working to Overcome the Global Impact of Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases. WHO, 2010Google Scholar,2.Tahir D. et al.Vector-borne nematode diseases in pets and humans in the Mediterranean Basin: an update.Vet. World. 2019; 12: 1630-1643Crossref PubMed Scopus (15) Google Scholar]. For example, DENV incidence has grown over 30-fold in the past 50 years, now reaching about 400 million cases per year [3.Bhatt S. et al.The global distribution and burden of dengue.Nature. 2013; 496: 504-507Crossref PubMed Scopus (5268) Google Scholar]. The recent ZIKV outbreak resulted in hundreds of thousands of infections and large-scale social and economic disruption [4.World Health Organization Zika Virus Outbreak Global Response: Interim Report. WHO, 2016Google Scholar]. While malaria cases are falling in southeast Asia, infections are rising in other parts of the world and remain 'unacceptably high' according to the World Health Organization [5.Guglielmi G. Malaria cases are falling worldwide.Nature. 2019; (Published online December 4, 2019. https://doi.org/10.1038/d41586-019-03746-3)Crossref Google Scholar]. Re-emergence and expansion of mosquito-borne diseases are due to many factors, including increased urbanization and global travel and trade, climate change, land use pattern changes, and unreliable piped water supply [6.World Health Organization Global Vector Control Response 2017-2030. WHO, 2017Google Scholar]. Current mosquito control strategies, including long-lasting insecticide-treated bed nets, chemical insecticides, and environmental management [7.Schreck C.E. Permethrin and dimethyl phthalate as tent fabric treatments against Aedes aegypti.J. Am. Mosq. Control Assoc. 1991; 7: 533-535PubMed Google Scholar], have been unable to address these diseases due to increasing genetic and behavioral vector resistance to these interventions [8.Succo T. et al.Autochthonous dengue outbreak in Nîmes, South of France, July to September 2015.Euro Surveill. 2016; 21: 21Crossref Scopus (0) Google Scholar]. In addition, chemical interventions have an unintended effect on important nontarget insects, such as pollinators [9.Ware G.W. Effects of pesticides on nontarget organisms.Residue Rev. 1980; 76: 173-201Crossref PubMed Scopus (49) Google Scholar]. Thus, new, more effective control strategies are urgently needed to address mosquito-borne diseases. In response to this growing need, the number of novel mosquito control technologies have expanded in recent years. Many of these involve the release of mosquitoes that aim to achieve population suppression (see Glossary) or population modification of wild type mosquitoes. Population suppression strategies aim to reduce or eliminate mosquito populations. Such strategies include sterile insect technique (SIT), incompatible insect technique (IIT), and transgenic-based technologies, where sterile insects mate with wild type insects and reduce population sizes in the next generation. Gene drive approaches, where alleles can increase in frequency over multiple generations, could potentially suppress populations after a single release or modify mosquitoes to be refractory or resistant to pathogens and prevent pathogen transmission. The release of mosquitoes carrying a symbiont or a gene drive into wild populations can enable the spread of the modification and result in entire populations becoming refractory to a pathogen. In this review, we summarize recent developments in the use of symbiont-infected mosquitoes and transgenic gene drive strategies, focusing on their different varieties and capabilities. The early symbiont-mediated mosquito [10.Laven H. Eradication of Culex pipiens fatigans through cytoplasmic incompatibility.Nature. 1967; 216: 383-384Crossref PubMed Scopus (275) Google Scholar] control was the introduction of nonmodified microorganisms into insects to reduce vector competence (Box 1). Wolbachia is the most extensively studied system for natural symbiont-based mosquito control. It may be the most common intracellular endosymbiont in arthropods and nematodes, with 60% of all insects harboring Wolbachia [11.Hilgenboecker K. et al.How many species are infected with Wolbachia?--A statistical analysis of current data.FEMS Microbiol. Lett. 2008; 281: 215-220Crossref PubMed Scopus (0) Google Scholar]. Wolbachia are transmitted vertically from mother to offspring and can maximize their transmission by manipulating host reproduction through feminization, parthenogenesis, male killing, and/or cytoplasmic incompatibility (CI). CI is induced when Wolbachia-infected males mate with uninfected females, which results in nonviable offspring. Wolbachia can inhibit or block infection with DENV, yellow fever, ZIKV, other arboviruses, and malaria parasite (Figure 1, Key figure ) [12.Bian G. et al.Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection.Science. 2013; 340: 748-751Crossref PubMed Scopus (289) Google Scholar, 13.Dutra H.L. et al.Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes.Cell Host Microbe. 2016; 19: 771-774Abstract Full Text Full Text PDF PubMed Google Scholar, 14.Hughes G.L. et al.Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae.PLoS Pathog. 2011; 7e1002043Crossref Scopus (214) Google Scholar]. Transfected or native Wolbachia infections have both been used for population suppression strategies [10.Laven H. Eradication of Culex pipiens fatigans through cytoplasmic incompatibility.Nature. 1967; 216: 383-384Crossref PubMed Scopus (275) Google Scholar,15.Zheng X. et al.Incompatible and sterile insect techniques combined eliminate mosquitoes.Nature. 2019; 572: 56-61Crossref PubMed Scopus (212) Google Scholar]. Interestingly, some important vector species, such as Aedes aegypti, are naturally free of Wolbachia [16.Gloria-Soria A. et al.Lack of evidence for natural Wolbachia infections in Aedes aegypti (Diptera: Culicidae).J. Med. Entomol. 2018; 55: 1354-1356PubMed Google Scholar,17.Ross P.A. et al.An elusive endosymbiont: does Wolbachia occur naturally in Aedes aegypti?.Ecol. Evol. 2020; 10: 1581-1591Crossref PubMed Scopus (27) Google Scholar], providing an open niche for infection. While there is conjecture if some of the major Anopheles vectors are truly infected [18.Chrostek E. Gerth M. Is Anopheles gambiae a natural host of Wolbachia?.mBio. 2019; 10e00784-19Crossref PubMed Scopus (24) Google Scholar], recent reports indicate other Anopheline species possess high-density native Wolbachia infections [19.Walker T. et al.Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes.Curr. Biol. 2021; 31: 2310-2320Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar]. This offers renewed promise for infection of medically relevant Anopheles vectors with these native strains that are adapted to the Anopheline environment. Several reviews have covered recent progress of Wolbachia-based mosquito control that exploit the bacterium [20.Flores H.A. O'Neill S.L. Controlling vector-borne diseases by releasing modified mosquitoes.Nat. Rev. Microbiol. 2018; 16: 508-518Crossref PubMed Scopus (145) Google Scholar, 21.Wang G.-H. et al.Combating mosquito-borne diseases using genetic control technologies.Nat. Commun. 2021; 12: 4388Crossref PubMed Scopus (0) Google Scholar, 22.Caragata E.P. et al.Wolbachia as translational science: controlling mosquito-borne pathogens.Trends Parasitol. 2021; 37: 1050-1067Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].Box 1Using symbionts as novel mosquito control strategiesThe increasingly emerging interactions among mosquito hosts, pathogen infection, and symbionts are inspiring the development of new strategies to exploit symbionts for vector-borne disease control [111.Gupta A. Nair S. Dynamics of insect-microbiome interaction influence host and microbial symbiont.Front. Microbiol. 2020; 11: 1357Crossref PubMed Scopus (25) Google Scholar]. Most importantly, symbiont-based mosquito control shows potential power to minimize the resistance problem and cause minimal side effects to the environment. The application of symbionts in vector control includes: (i) delivering natural symbionts into the mosquito directly to disrupt mosquito physiology to reduce vector competence or display antipathogen effects; (ii) genetic modification of symbionts to express antipathogen effector molecules, then delivering the engineered symbiont into the mosquito so that the mosquito is resistant to pathogen or there is decreased vector competence [112.Gao H. et al.Mosquito microbiota and implications for disease control.Trends Parasitol. 2020; 36: 98-111Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar] or vectorial capacity [113.Cansado-Utrilla C. et al.The microbiome and mosquito vectorial capacity: rich potential for discovery and translation.Microbiome. 2021; 9: 111Crossref PubMed Scopus (9) Google Scholar] (Figure I).Figure 1Key figure. Symbiont-based approaches for mosquito control.Show full caption(A) Natural bacteria-based approaches for mosquito control. Wolbachia-infected males can suppress mosquito populations through cytoplasmic incompatibility (CI) effects or Wolbachia-infected mosquitoes modify mosquito populations for pathogen resistance. Serratia Y1 and Asaia induce the mosquito immunity system and slow the malaria parasite development. The secondary metabolites of Serratia ureilytica Su_YN1 and Chromobacterium can be responsible for antiplasmodial or dengue virus (DENV)-2. (B) Natural fungus-based approaches for mosquito control. Wickerhamomyces anomalus can be used against malaria parasite development through secreting toxin protein and Beauveria bassiana can be used against DENV-2 and Zika virus (ZIKV) activity through inducing the mosquito immunity system. (C) Natural insect-specific viruses (Eilat virus, cell fusing agent virus, Phasi Charoen-like virus, and Negevirus) can inhibit arbovirus development, either alone or in combination. (D) Engineered bacteria-based approaches for mosquito control. Engineered Serratia AS1 and Asaia can express antiplasmodial effector proteins to inhibit Plasmodium development. Engineered Asaia can induce mosquito immunity to control parasite Dirofilaria immitis. (E) Engineered fungus-based approaches for mosquito control. Engineered Metarhizium pingshaense-infected mosquito has shorter life spans and reproductive output than wild type mosquitoes. (F) Engineered virus-based approaches for mosquito control. Engineered Negevirus expressed an anti-chikungunya virus (CHIKV) antibody that can inhibit the CHIKV replication.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increasingly emerging interactions among mosquito hosts, pathogen infection, and symbionts are inspiring the development of new strategies to exploit symbionts for vector-borne disease control [111.Gupta A. Nair S. Dynamics of insect-microbiome interaction influence host and microbial symbiont.Front. Microbiol. 2020; 11: 1357Crossref PubMed Scopus (25) Google Scholar]. Most importantly, symbiont-based mosquito control shows potential power to minimize the resistance problem and cause minimal side effects to the environment. The application of symbionts in vector control includes: (i) delivering natural symbionts into the mosquito directly to disrupt mosquito physiology to reduce vector competence or display antipathogen effects; (ii) genetic modification of symbionts to express antipathogen effector molecules, then delivering the engineered symbiont into the mosquito so that the mosquito is resistant to pathogen or there is decreased vector competence [112.Gao H. et al.Mosquito microbiota and implications for disease control.Trends Parasitol. 2020; 36: 98-111Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar] or vectorial capacity [113.Cansado-Utrilla C. et al.The microbiome and mosquito vectorial capacity: rich potential for discovery and translation.Microbiome. 2021; 9: 111Crossref PubMed Scopus (9) Google Scholar] (Figure I). (A) Natural bacteria-based approaches for mosquito control. Wolbachia-infected males can suppress mosquito populations through cytoplasmic incompatibility (CI) effects or Wolbachia-infected mosquitoes modify mosquito populations for pathogen resistance. Serratia Y1 and Asaia induce the mosquito immunity system and slow the malaria parasite development. The secondary metabolites of Serratia ureilytica Su_YN1 and Chromobacterium can be responsible for antiplasmodial or dengue virus (DENV)-2. (B) Natural fungus-based approaches for mosquito control. Wickerhamomyces anomalus can be used against malaria parasite development through secreting toxin protein and Beauveria bassiana can be used against DENV-2 and Zika virus (ZIKV) activity through inducing the mosquito immunity system. (C) Natural insect-specific viruses (Eilat virus, cell fusing agent virus, Phasi Charoen-like virus, and Negevirus) can inhibit arbovirus development, either alone or in combination. (D) Engineered bacteria-based approaches for mosquito control. Engineered Serratia AS1 and Asaia can express antiplasmodial effector proteins to inhibit Plasmodium development. Engineered Asaia can induce mosquito immunity to control parasite Dirofilaria immitis. (E) Engineered fungus-based approaches for mosquito control. Engineered Metarhizium pingshaense-infected mosquito has shorter life spans and reproductive output than wild type mosquitoes. (F) Engineered virus-based approaches for mosquito control. Engineered Negevirus expressed an anti-chikungunya virus (CHIKV) antibody that can inhibit the CHIKV replication. Besides Wolbachia, research in other natural symbionts for mosquito control has made rapid progress recently (Figure 1A,B). Serratia Y1 bacteria from field-caught Anopheles sinensis can inhibit Plasmodium berghei by modulating mosquito immunity genes to inhibit Plasmodium development [23.Bai L. et al.A gut symbiotic bacterium Serratia marcescens renders mosquito resistance to Plasmodium infection through activation of mosquito immune responses.Front. Microbiol. 2019; 10: 1580Crossref PubMed Scopus (0) Google Scholar]. Asaia bacteria can interact with the Anopheles mosquito immune system to slow malaria parasite development [24.Cappelli A. et al.Asaia activates immune genes in mosquito eliciting an anti-Plasmodium response: implications in malaria control.Front. Genet. 2019; 10: 836Crossref PubMed Scopus (20) Google Scholar]. Symbionts can not only interact with mosquitoes to interfere with pathogens, but they can also inhibit pathogens directly. For example, Serratia ureilytica Su_YN1 directly secretes an antimalarial lipase that kills Plasmodium parasites at different stages, effectively preventing parasite infection [25.Gao H. et al.A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase.Nat. Microbiol. 2021; 6: 806-817Crossref PubMed Scopus (6) Google Scholar]. The symbiont can also show antipathogen activity by their secondary metabolites [26.Wu P. et al.A gut commensal bacterium promotes mosquito permissiveness to arboviruses.Cell Host Microbe. 2018; 25: 101-112Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Likewise, Chromobacterium inhibits other midgut bacteria growth and displays entomopathogenic activity to mosquito larvae and adults. Romidepsin might be the Chromobacterium secondary metabolite responsible for the antiplasmodial activity [27.Saraiva R.G. et al.Chromobacterium spp. mediate their anti-Plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin.Sci. Rep. 2018; 8: 6176Crossref PubMed Scopus (23) Google Scholar]; the Chromobacterium secondary metabolite aminopeptidase interferes with DENV-2 attachment by increasing the degradation of the Flavivirus E protein [28.Saraiva R.G. et al.Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.PLoS Negl. Trop. Dis. 2018; 12e0006443Crossref Scopus (27) Google Scholar]. Natural symbiotic fungi also show potential for mosquito-borne disease control. Wickerhamomyces anomalus is a yeast that secretes a killer toxin protein that shows strong activity against P. berghei at different developmental stages [29.Cappelli A. et al.Killer yeasts exert anti-plasmodial activities against the malaria parasite Plasmodium berghei in the vector mosquito Anopheles stephensi and in mice.Parasite Vector. 2019; 12: 329Crossref PubMed Scopus (0) Google Scholar]. Beauveria bassiana induces the Toll/Jak-Stat immune pathways and reduces mosquito vector competence for DENV-2 in A. aegypti [30.Dong Y. et al.The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti.Insect Biochem. Mol. Biol. 2012; 42: 126-132Crossref PubMed Scopus (80) Google Scholar] and Aedes albopictus capacity for ZIKV [31.Deng S.Q. et al.Beauveria bassiana infection reduces the vectorial capacity of Aedes albopictus for the Zika virus.J. Pest. Sci. 2019; 92: 781-789Crossref Scopus (8) Google Scholar]. Beyond bacteria and fungi, insect-specific viruses (ISVs) also can be used to control arboviruses. Cell fusing agent virus (CFAV) is the early recognized ISV from A. aegypti cells that can cause cell fusing phenotype in A. albopictus cells [32.Stollar V. Thomas V.L. An agent in the Aedes aegypti cell line (Peleg) which causes fusion of Aedes albopictus cells.Virology. 1975; 64: 367-377Crossref PubMed Scopus (167) Google Scholar]. Another mosquito ISV, Eilat virus (EILV), can reduce CHIKV titers and delay replication in vitro. When A. aegypti mosquitoes were infected with EILV, dissemination of CHIKV was delayed by a heterologous interference mechanism [33.Nasar F. et al.Eilat virus induces both homologous and heterologous interference.Am. J. Trop. Med. Hyg. 2015; 93: 226Google Scholar]. Co-infection of different ISVs also can inhibit arbovirus development. For example, CFAV and Phasi Charoen-like virus co-infection can inhibit the growth of ZIKV and DENV in A. albopictus cells [34.Schultz M.J. et al.Dual insect specific virus infection limits arbovirus replication in Aedes mosquito cells.Virology. 2018; 518: 406-413Crossref PubMed Scopus (46) Google Scholar]. Negevirus is another recently discovered ISV [35.Vasilakis N. et al.Negevirus: a proposed new taxon of insect-specific viruses with wide geographic distribution.J. Virol. 2013; 87: 2475-2488Crossref PubMed Scopus (121) Google Scholar]. When Negevirus infected A. albopictus cells, the cells could not be infected with CHIKV and Mayaro viruses [36.Patterson E.I. et al.Negeviruses reduce replication of alphaviruses during coinfection.J. Virol. 2021; 95e0043321Crossref PubMed Scopus (2) Google Scholar]. All these results suggest that ISVs can be potential tools to control arboviruses through superinfection exclusion, which needs further testing in mosquito population. Engineered symbionts producing antipathogen or immunomodulatory effector molecules (termed paratransgenesis) is another powerful symbiont-mediated mosquito control approach (Figure 1C,D). After the symbiont is engineered, it is reintroduced into the arthropod host to reduce its vector competence (Box 1). There are some critical requirements for the candidate symbiont. First, the symbiont should be able to stably spread into the population vertically and/or horizontally and maintain in the population long enough to express the effector molecules [37.Wilke A.B. Marrelli M.T. Paratransgenesis: a promising new strategy for mosquito vector control.Parasit. Vectors. 2015; 8: 342Crossref PubMed Scopus (97) Google Scholar]. Second, the symbiont should be easily culturable and genetically manipulatable, while not reducing the host fitness [38.Lovett B. et al.Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso.Science. 2019; 364: 894-897Crossref PubMed Scopus (40) Google Scholar]. Third, the symbiont should express the effector molecules to interfere with the target pathogen [39.Wang S. et al.Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria.Science. 2017; 357: 1399-1402Crossref PubMed Scopus (116) Google Scholar]. There are several candidates that have shown potential attributes to be a paratransgenesis symbiont. Serratia (AS1), which was isolated from Anopheles ovaries, can be transmitted vertically and horizontally, facilitating its spread into mosquito populations. Furthermore, the genetically engineered AS1 can express anti-Plasmodium effector proteins that inhibit Plasmodium development in mosquitoes [39.Wang S. et al.Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria.Science. 2017; 357: 1399-1402Crossref PubMed Scopus (116) Google Scholar]. Together, this suggests that AS1 is a promising candidate for Plasmodium control. Asaia bogorensis is transmitted vertically and can populate the larval and adult gut and reproductive organs of Anopheles and Aedes mosquitoes [40.Favia G. et al.Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9047-9051Crossref PubMed Scopus (296) Google Scholar]. Recently, Asaia was successfully engineered to conditionally express the anti-plasmodial protein scorpine, which significantly inhibits the development of malaria parasites, while displaying a reduced fitness cost compared with an Asaia stain constitutively expressing the antiplasmodial effector [41.Shane J.L. et al.Blood meal-induced inhibition of vector-borne disease by transgenic microbiota.Nat. Commun. 2018; 9: 4127Crossref PubMed Scopus (31) Google Scholar]. More recently, Asaia was engineered to induce an immune response within Aedes and Anopheles mosquitoes to control the heartworm parasite Dirofilaria immitis [42.Epis S. et al.Chimeric symbionts expressing a Wolbachia protein stimulate mosquito immunity and inhibit filarial parasite development.Commun. Biol. 2020; 3: 105Crossref PubMed Scopus (8) Google Scholar]. Notably, both engineered AS1 and Asaia can be spread into mosquito populations and keep the antipathogen capability in the laboratory or semi-field conditions [39.Wang S. et al.Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria.Science. 2017; 357: 1399-1402Crossref PubMed Scopus (116) Google Scholar]. Intriguingly, Wolbachia and Asaia appear antagonistic to one another. Wolbachia-infected mosquito showed lower Asaia densities compared with their uninfected counterparts, while removing Asaia from Anopheles mosquitoes enabled vertical transmission of Wolbachia [43.Rosso F. et al.Reduced diversity of gut microbiota in two Aedes mosquitoes species in areas of recent invasion.Sci. Rep. 2018; 8: 16091Crossref PubMed Scopus (20) Google Scholar,44.Hughes G.L. et al.Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 12498-12503Crossref PubMed Scopus (137) Google Scholar]. Genetically engineered Metarhizium pingshaense (Mp-hybrid) infection of Anopheles coluzzii had shorter lifespans and reproductive output compared with wild type mosquitoes. Furthermore, Mp-hybrid showed higher virulence and lower inoculum load than wild type fungus in a semi-field trial in Burkina Faso [38.Lovett B. et al.Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso.Science. 2019; 364: 894-897Crossref PubMed Scopus (40) Google Scholar]. Finally, modifying the recently discovered ISV, Negevirus, to express anti-CHIKV antibodies inhibited CHIKV replication [36.Patterson E.I. et al.Negeviruses reduce replication of alphaviruses during coinfection.J. Virol. 2021; 95e0043321Crossref PubMed Scopus (2) Google Scholar]. All these results indicate that engineered bacteria, fungus, and even viruses can be used directly or combined with existing chemical control strategies for mosquito control. The genetic basis of CI in Wolbachia has recently been identified by compelling evidence that two genes, cifA and cifB, are involved in induction and rescue. While the specific models and mechanism(s) of CI still remain to be elucidated, it has been shown that expression of these bacterial genes in the host germline can recapitulate the CI phenotype. Expression of cifA in females rescues CI, while intriguingly, it appears that coexpression of both cifA and cifB in males is required to induce this phenotype [45.Beckmann J.F. et al.A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility.Nat. Microbiol. 2017; 2: 17007Crossref PubMed Scopus (181) Google Scholar,46.LePage D.P. et al.Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility.Nature. 2017; 543: 243-247Crossref PubMed Scopus (218) Google Scholar]. 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