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Manipulating Mosquito Tolerance for Arbovirus Control

2019; Cell Press; Volume: 26; Issue: 3 Linguagem: Inglês

10.1016/j.chom.2019.08.005

1934-6069

Louis Lambrechts, María-Carla Saleh,

Insect-Plant Interactions and Control

The inexorable emergence of mosquito-borne arboviruses and the failure of traditional vector control methods to prevent their transmission have triggered the development of alternative entomological interventions to render mosquito populations incapable of carrying arboviruses. Here, we use a theoretical framework to argue that decreasing mosquito tolerance to arbovirus infection could be a more evolutionarily sustainable disease control strategy than increasing mosquito resistance. Increasing resistance is predicted to select for mutant arboviruses escaping resistance, whereas reducing tolerance should lead to the death of infected vectors and thus select for mosquito-attenuated arbovirus variants that are less transmissible. The inexorable emergence of mosquito-borne arboviruses and the failure of traditional vector control methods to prevent their transmission have triggered the development of alternative entomological interventions to render mosquito populations incapable of carrying arboviruses. Here, we use a theoretical framework to argue that decreasing mosquito tolerance to arbovirus infection could be a more evolutionarily sustainable disease control strategy than increasing mosquito resistance. Increasing resistance is predicted to select for mutant arboviruses escaping resistance, whereas reducing tolerance should lead to the death of infected vectors and thus select for mosquito-attenuated arbovirus variants that are less transmissible. In every host-pathogen relationship, the host can survive by employing two conceptually different strategies. It can resist the infection, or it can limit—tolerate—the deleterious effects of the infection. Distinction between these two defense strategies was made as early as 1932 by Clunies-Ross in the context of gastro-intestinal parasites affecting sheep farming (Clunies-Ross, 1932Clunies-Ross I. Observations on the resistance of sheep to infestations by the stomach worm, Haemonchus contortus.J. Sci. Ind. Res. 1932; 5: 73-80Google Scholar). He proposed that a breeding program to minimize production losses due to infection would be more profitable than a breeding program to increase parasite resistance per se. This concept was subsequently revisited in the 1980s by Albers, who distinguished "resistance," defined as the ability to suppress establishment and/or subsequent development of infection, from "resilience," defined as the ability to maintain a relatively undepressed production level when infected (Albers et al., 1987Albers G.A. Gray G.D. Piper L.R. Barker J.S. Le Jambre L.F. Barger I.A. The genetics of resistance and resilience to Haemonchus contortus infection in young merino sheep.Int. J. Parasitol. 1987; 17: 1355-1363Crossref PubMed Scopus (231) Google Scholar). In the 1990s, plant ecologists resurfaced the conceptual framework of a two-faceted defense response relying on host resistance—the ability to limit pathogen burden—and host tolerance—the ability to limit the disease severity induced by a given pathogen burden (Simms and Triplett, 1994Simms E.L. Triplett J. Costs and benefits of plant responses to disease: resistance and tolerance.Evolution. 1994; 48: 1973-1985Crossref PubMed Google Scholar). In this literature, tolerance is usually defined as the slope of host fitness against infection intensity. In the 2000s, animal parasitologists borrowed this approach to measure tolerance (the reaction norm of host health to pathogen burden) in a rodent malaria model (Råberg et al., 2007Råberg L. Sim D. Read A.F. Disentangling genetic variation for resistance and tolerance to infectious diseases in animals.Science. 2007; 318: 812-814Crossref PubMed Scopus (534) Google Scholar). This landmark study demonstrated the existence of genetic variation in tolerance and a genetic trade-off between resistance and tolerance in an animal host-pathogen system. Genetic variation is a prerequisite for a trait to evolve in response to selective pressure. Because resistance has a negative effect on pathogen fitness whereas tolerance does not, their relative importance, and the extent to which their genetic basis is shared, can result in a variety of evolutionary trajectories for both the host and the pathogen (Svensson and Råberg, 2010Svensson E.I. Råberg L. Resistance and tolerance in animal enemy-victim coevolution.Trends Ecol. Evol. 2010; 25: 267-274Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Following these seminal studies, a mathematical framework has been developed based on empirical data from several model organisms (including the fruit fly and the mouse) to distinguish between tolerance and resistance mechanisms, with the hope of improving treatment and prevention of infectious disease in humans and animals (Medzhitov et al., 2012Medzhitov R. Schneider D.S. Soares M.P. Disease tolerance as a defense strategy.Science. 2012; 335: 936-941Crossref PubMed Scopus (1048) Google Scholar, Schneider and Ayres, 2008Schneider D.S. Ayres J.S. Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases.Nat. Rev. Immunol. 2008; 8: 889-895Crossref PubMed Scopus (0) Google Scholar). In this mini review, we introduce the idea of manipulating the tolerance of mosquito vectors for the control of human diseases caused by mosquito-borne arboviruses, such as dengue, Zika, and chikungunya (Wilder-Smith et al., 2017Wilder-Smith A. Gubler D.J. Weaver S.C. Monath T.P. Heymann D.L. Scott T.W. Epidemic arboviral diseases: priorities for research and public health.Lancet Infect. Dis. 2017; 17: e101-e106Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Hereafter, we use the term tolerance to refer to all host defense mechanisms that limit "damage to functions and structures" during infection, without interfering with pathogen load, as was defined more than 60 years ago by plant pathologists (Caldwell et al., 1958Caldwell R.M. Schafer J.F. Compton L.E. Patterson F.L. Tolerance to cereal leaf rusts.Science. 1958; 128: 714-715Crossref PubMed Scopus (74) Google Scholar). The failure of current methods for mosquito-borne arbovirus control has stimulated the development of several innovative strategies (Achee et al., 2019Achee N.L. Grieco J.P. Vatandoost H. Seixas G. Pinto J. Ching-Ng L. Martins A.J. Juntarajumnong W. Corbel V. Gouagna C. et al.Alternative strategies for mosquito-borne arbovirus control.PLoS Negl. Trop. Dis. 2019; 13: e0006822PubMed Google Scholar). One of these strategies, referred to as population modification, aims at rendering the wild vector population incapable of transmitting pathogens (Flores and O'Neill, 2018Flores H.A. O'Neill S.L. Controlling vector-borne diseases by releasing modified mosquitoes.Nat. Rev. Microbiol. 2018; 16: 508-518Crossref PubMed Scopus (163) Google Scholar). To date, however, the development of novel interventions based on the population modification approach has focused on increasing mosquito resistance to arbovirus infection through, for example, introgression of antiviral transgenes or protective Wolbachia endosymbionts (Kean et al., 2015Kean J. Rainey S.M. McFarlane M. Donald C.L. Schnettler E. Kohl A. Pondeville E. Fighting arbovirus transmission: natural and engineered control of vector competence in Aedes mosquitoes.Insects. 2015; 6: 236-278Crossref PubMed Scopus (51) Google Scholar). One of the main challenges to the sustainability of these strategies is arbovirus evolution (Bull and Turelli, 2013Bull J.J. Turelli M. Wolbachia versus dengue: evolutionary forecasts.Evol. Med. Public Health. 2013; 2013: 197-207Crossref PubMed Google Scholar). Killing a pathogen and/or reducing its growth are expected to strongly select for pathogen escape mutants, which can quickly undermine control. Conversely, strategies generating weaker selection for pathogen countermeasures are considered more evolutionarily robust or "evolution-proof" (Allen et al., 2014Allen R.C. Popat R. Diggle S.P. Brown S.P. Targeting virulence: can we make evolution-proof drugs?.Nat. Rev. Microbiol. 2014; 12: 300-308Crossref PubMed Scopus (354) Google Scholar). Here, we argue that decreasing mosquito tolerance to arbovirus infection—leading to the death of infected vectors—could be a more evolutionarily sustainable arbovirus control strategy than increasing mosquito resistance (Figure 1). Unlike arboviral infections in humans, which are usually acute, arboviral infections in mosquitoes are persistent. Once the infection is established, the mosquito remains infected for the rest of its life. Despite active replication and high viral loads, however, arboviral infections in mosquitoes typically do not result in severe fitness defects. Although some fitness costs of infection have been reported (e.g., da Silveira et al., 2018da Silveira I.D. Petersen M.T. Sylvestre G. Garcia G.A. David M.R. Pavan M.G. Maciel-de-Freitas R. Zika virus infection produces a reduction on Aedes aegypti lifespan but no effects on mosquito fecundity and oviposition success.Front. Microbiol. 2018; 9: 3011Crossref PubMed Scopus (11) Google Scholar, Grimstad et al., 1980Grimstad P.R. Ross Q.E. Craig Jr., G.B. Aedes triseriatus (Diptera: Culicidae) and la Crosse virus. II. Modification of mosquito feeding behavior by virus infection.J. Med. Entomol. 1980; 17: 1-7Crossref PubMed Scopus (89) Google Scholar, Styer et al., 2007Styer L.M. Meola M.A. Kramer L.D. West Nile virus infection decreases fecundity of Culex tarsalis females.J. Med. Entomol. 2007; 44: 1074-1085Crossref PubMed Scopus (48) Google Scholar), many other studies have failed to detect experimental evidence for arbovirus virulence in mosquitoes (e.g., Ciota et al., 2011Ciota A.T. Styer L.M. Meola M.A. Kramer L.D. The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes.BMC Ecol. 2011; 11: 23Crossref PubMed Scopus (28) Google Scholar). Overall, the cost of infection is usually modest and context-dependent (Lambrechts and Scott, 2009Lambrechts L. Scott T.W. Mode of transmission and the evolution of arbovirus virulence in mosquito vectors.Proc. Biol. Sci. 2009; 276: 1369-1378Crossref PubMed Scopus (97) Google Scholar), and mosquitoes are considered tolerant to arbovirus infections. This is particularly remarkable for arboviruses such as yellow fever virus, which causes substantial mortality in immunologically naïve humans but not in their mosquito vectors (Barrett and Higgs, 2007Barrett A.D. Higgs S. Yellow fever: a disease that has yet to be conquered.Annu. Rev. Entomol. 2007; 52: 209-229Crossref PubMed Scopus (266) Google Scholar). Mosquito tolerance is essential for arbovirus fitness because both high viral loads and mosquito survival are necessary for successful arbovirus transmission to the human host. How do mosquitoes cope with viral infection? Response to infection does not simply consist of activating immune pathways, it also encompasses a broad range of physiological consequences including metabolic adaptations, stress responses, and tissue repair. Critically, upon infection, the homeostatic regulation of these pathways is altered. However, such alterations do not always result in increased disease severity or acute infections but can also lead to improved survival (or health) despite active pathogen replication, which defines tolerance. A clear example of this conundrum can be found in Drosophila, where the transcription factor CrebA is induced upon bacterial infection (Troha et al., 2018Troha K. Im J.H. Revah J. Lazzaro B.P. Buchon N. Comparative transcriptomics reveals CrebA as a novel regulator of infection tolerance in D. melanogaster.PLoS Pathog. 2018; 14: e1006847Crossref PubMed Scopus (53) Google Scholar). Interestingly, CrebA-deficient flies are more likely to die from infection despite carrying the same number of bacteria as wildtype flies. CrebA is expressed in the fat body, an organ analogous to the mammalian liver and adipose tissues (Søndergaard, 1993Søndergaard L. Homology between the mammalian liver and the Drosophila fat body.Trends Genet. 1993; 9: 193Abstract Full Text PDF PubMed Scopus (72) Google Scholar), where it regulates the transcription of multiple secretory pathway genes. Loss of CrebA during infection triggers endoplasmic reticulum stress, which is sufficient to sensitize flies to infection. These results suggest that immune tolerance modulates host physiology to prevent the deleterious effect of infection-associated cellular stress. There is increasing evidence to suggest that viral infections in insects cause intracellular stress and that the cellular management of stress favors virus replication. For example, dengue virus replication in mosquito cells is promoted by activation of the PERK pathway during endoplasmic reticulum stress (Hou et al., 2017Hou J.N. Chen T.H. Chiang Y.H. Peng J.Y. Yang T.H. Cheng C.C. Sofiyatun E. Chiu C.H. Chiang-Ni C. Chen W.J. PERK signal-modulated protein translation promotes the survivability of dengue 2 virus-infected mosquito cells and extends viral replication.Viruses. 2017; 9Crossref PubMed Scopus (15) Google Scholar), autophagy (Brackney, 2017Brackney D.E. Implications of autophagy on arbovirus infection of mosquitoes.Curr. Opin. Insect Sci. 2017; 22: 1-6Crossref PubMed Scopus (11) Google Scholar), and oxidative stress (Chen et al., 2012Chen T.H. Lo Y.P. Yang C.F. Chen W.J. Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection.PLoS Negl. Trop. Dis. 2012; 6: e1613Crossref PubMed Scopus (19) Google Scholar, Chen et al., 2011Chen T.H. Tang P. Yang C.F. Kao L.H. Lo Y.P. Chuang C.K. Shih Y.T. Chen W.J. Antioxidant defense is one of the mechanisms by which mosquito cells survive dengue 2 viral infection.Virology. 2011; 410: 410-417Crossref PubMed Scopus (56) Google Scholar, and Chen et al., 2017Chen T.H. Chiang Y.H. Hou J.N. Cheng C.C. Sofiyatun E. Chiu C.H. Chen W.J. XBP1-mediated BiP/GRP78 upregulation copes with oxidative stress in mosquito cells during dengue 2 virus infection.BioMed Res. Int. 2017; 2017: 3519158Crossref PubMed Scopus (20) Google Scholar, Riahi et al., 2019Riahi H. Brekelmans C. Foriel S. Merkling S.H. Lyons T.A. Itskov P.M. Kleefstra T. Ribeiro C. van Rij R.P. Kramer J.M. et al.The histone methyltransferase G9a regulates tolerance to oxidative stress-induced energy consumption.PLoS Biol. 2019; 17: e2006146Crossref PubMed Scopus (17) Google Scholar). Cell homeostasis in regulating infection can thus be tuned by both the virus and the immune system. Pathogens often manipulate the behavior of their host in order to enhance their own transmission (Hughes and Libersat, 2019Hughes D.P. Libersat F. Parasite manipulation of host behavior.Curr. Biol. 2019; 29: R45-R47Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Such behavioral manipulations have been widely observed in mosquito vectors of human pathogens (Hurd, 2003Hurd H. Manipulation of medically important insect vectors by their parasites.Annu. Rev. Entomol. 2003; 48: 141-161Crossref PubMed Scopus (236) Google Scholar, Lefèvre and Thomas, 2008Lefèvre T. Thomas F. Behind the scene, something else is pulling the strings: emphasizing parasitic manipulation in vector-borne diseases.Infect. Genet. Evol. 2008; 8: 504-519Crossref PubMed Scopus (133) Google Scholar). Behavioral changes of infected vectors are often deviations in the degree and timing of normal behaviors, including host seeking, host attack persistence, and blood-feeding efficiency (Murdock et al., 2017Murdock C.C. Luckhart S. Cator L.J. Immunity, host physiology, and behaviour in infected vectors.Curr. Opin. Insect Sci. 2017; 20: 28-33Crossref PubMed Scopus (14) Google Scholar). For example, Aedes triseriatus females infected by LaCrosse virus tend to probe more, engorge less, and re-feed more often than non-infected siblings (Grimstad et al., 1980Grimstad P.R. Ross Q.E. Craig Jr., G.B. Aedes triseriatus (Diptera: Culicidae) and la Crosse virus. II. Modification of mosquito feeding behavior by virus infection.J. Med. Entomol. 1980; 17: 1-7Crossref PubMed Scopus (89) Google Scholar, Jackson et al., 2012Jackson B.T. Brewster C.C. Paulson S.L. La Crosse virus infection alters blood feeding behavior in Aedes triseriatus and Aedes albopictus (Diptera: Culicidae).J. Med. Entomol. 2012; 49: 1424-1429Crossref PubMed Scopus (35) Google Scholar). In some cases, however, the arbovirus-induced behavioral changes in mosquitoes seem to reduce virus transmission potential. For example, West Nile virus infection reduced host-seeking activity of Culex pipiens females and did not induce a shift in their host preference toward birds (Vogels et al., 2017Vogels C.B.F. Fros J.J. Pijlman G.P. van Loon J.J.A. Gort G. Koenraadt C.J.M. Virus interferes with host-seeking behaviour of mosquito.J. Exp. Biol. 2017; 220: 3598-3603Crossref PubMed Scopus (21) Google Scholar). In those cases, behavioral changes are likely side effects resulting from immune responses and infection-related alterations in host physiology (Murdock et al., 2017Murdock C.C. Luckhart S. Cator L.J. Immunity, host physiology, and behaviour in infected vectors.Curr. Opin. Insect Sci. 2017; 20: 28-33Crossref PubMed Scopus (14) Google Scholar). To the best of our knowledge, no study to date has examined the possible manipulation of mosquito tolerance mechanisms by arboviruses. It would seem advantageous for an arbovirus to promote mosquito tolerance to infection, which would allow high viral loads (and therefore high transmission rate) without compromising mosquito survival (and therefore extending transmission duration). An important first step to address this question is to elucidate the molecular mechanisms of tolerance. Recent studies to decipher tolerance mechanisms in animals encompass various experimental models, including Drosophila infection by bacteria (Ayres et al., 2008Ayres J.S. Freitag N. Schneider D.S. Identification of Drosophila mutants altering defense of and endurance to Listeria monocytogenes infection.Genetics. 2008; 178: 1807-1815Crossref PubMed Scopus (93) Google Scholar) or RNA viruses (Merkling et al., 2015Merkling S.H. Bronkhorst A.W. Kramer J.M. Overheul G.J. Schenck A. Van Rij R.P. The epigenetic regulator G9a mediates tolerance to RNA virus infection in Drosophila.PLoS Pathog. 2015; 11: e1004692Crossref PubMed Scopus (60) Google Scholar, Teixeira et al., 2008Teixeira L. Ferreira A. Ashburner M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster.PLoS Biol. 2008; 6: e2Crossref PubMed Scopus (801) Google Scholar), mouse infection by Trypanosoma brucei (Olivera et al., 2016Olivera G.C. Ren X. Vodnala S.K. Lu J. Coppo L. Leepiyasakulchai C. Holmgren A. Kristensson K. Rottenberg M.E. Nitric oxide protects against infection-induced neuroinflammation by preserving the stability of the blood-brain barrier.PLoS Pathog. 2016; 12: e1005442Crossref PubMed Scopus (46) Google Scholar), Plasmodium chabaudi (Råberg et al., 2007Råberg L. Sim D. Read A.F. Disentangling genetic variation for resistance and tolerance to infectious diseases in animals.Science. 2007; 318: 812-814Crossref PubMed Scopus (534) Google Scholar) or influenza virus (Furuya et al., 2015Furuya Y. Furuya A.K. Roberts S. Sanfilippo A.M. Salmon S.L. Metzger D.W. Prevention of influenza virus-induced immunopathology by TGF-beta produced during allergic asthma.PLoS Pathog. 2015; 11: e1005180Crossref PubMed Scopus (30) Google Scholar), and monarch butterfly infection by protozoan parasites (Altizer et al., 2015Altizer S. Hobson K.A. Davis A.K. De Roode J.C. Wassenaar L.I. Do healthy monarchs migrate farther? Tracking natal origins of parasitized vs. uninfected monarch butterflies overwintering in Mexico.PLoS One. 2015; 10: e0141371Crossref PubMed Scopus (60) Google Scholar). Although significant progress has been made, the genes and processes that control tolerance remain elusive. Besides canonical immune pathways, various molecular pathways that are activated upon infection could contribute to tolerance to infection. In a recent study, we showed that mosquito tolerance to arbovirus infection depends on cellular reverse transcriptase (RT) activity (Goic et al., 2016Goic B. Stapleford K.A. Frangeul L. Doucet A.J. Gausson V. Blanc H. Schemmel-Jofre N. Cristofari G. Lambrechts L. Vignuzzi M. et al.Virus-derived DNA drives mosquito vector tolerance to arboviral infection.Nat. Commun. 2016; 7: 12410Crossref PubMed Scopus (134) Google Scholar). Fragments of RNA viruses such as dengue and chikungunya viruses are reverse transcribed by endogenous RT activity, and the resulting viral DNA is necessary for mosquito tolerance. Preventing viral DNA synthesis with the RT inhibitor azidothymidine (AZT) results in reduced mosquito survival without significant changes in viral loads (Figure 2). Evidence of mosquito death following arbovirus infection in the absence of viral DNA provides the proof of principle that infected mosquitoes can be selectively eliminated through the loss of tolerance. The rationale behind reducing mosquito tolerance rather than increasing resistance to arbovirus infection is that loss of tolerance is expected to be more evolutionarily sustainable. According to evolutionary theory, reducing tolerance should select for arbovirus variants that are less virulent to their vector (i.e., attenuated). This is in contrast with increasing resistance, which is predicted to select for arbovirus variants that are more virulent to their vectors. When mosquito tolerance is reduced, arbovirus attenuation results from a lower evolutionary optimum for viral load, which is also predicted to result in lower transmissibility due to the expected positive relationship between viral load and transmission rate (Figure 3). The evolutionary response to an intervention that targets mosquito-arbovirus interactions can be examined from both the mosquito and the arbovirus perspectives. There are two reasons for which the evolutionary response of mosquitoes is unlikely to undermine an intervention due to the arbovirus. First, as mentioned above, the deleterious effects of arbovirus infection are generally modest, and the mosquito fitness is largely maintained. Second, the prevalence of arbovirus infection in natural mosquito populations is exceedingly low. For example, dengue virus was only detected in 0.1% of Aedes aegypti mosquitoes randomly collected in a highly endemic region of Thailand (Yoon et al., 2012Yoon I.K. Getis A. Aldstadt J. Rothman A.L. Tannitisupawong D. Koenraadt C.J. Fansiri T. Jones J.W. Morrison A.C. Jarman R.G. et al.Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages.PLoS Negl. Trop. Dis. 2012; 6: e1730Crossref PubMed Scopus (106) Google Scholar). The prevalence reached about 1% when mosquitoes were collected in households with evidence of recent dengue virus transmission (Yoon et al., 2012Yoon I.K. Getis A. Aldstadt J. Rothman A.L. Tannitisupawong D. Koenraadt C.J. Fansiri T. Jones J.W. Morrison A.C. Jarman R.G. et al.Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages.PLoS Negl. Trop. Dis. 2012; 6: e1730Crossref PubMed Scopus (106) Google Scholar). In light of this, it is reasonable to suggest that arboviruses do not represent a major selective pressure driving the evolution of mosquito populations in natural settings. Thus, manipulation of mosquito resistance and tolerance to arbovirus infection should not lead to significant selection or counter-selection due to the arbovirus selective pressure. For instance, it means that the death of arbovirus-infected mosquitoes following the loss of tolerance is unlikely to drive the evolution of mosquitoes to restore higher tolerance. The evolutionary response that deserves more attention is that of the arbovirus. Because transmission by mosquitoes is an essential step of the viral lifecycle, any intervention that modifies mosquito-arbovirus interactions to reduce transmission may select for arbovirus mutants that escape or counteract the mechanism of the intervention as a reduction of transmission directly translates into a decrease in arbovirus fitness. In the case of increased mosquito resistance by genetic engineering or Wolbachia transfection (Kean et al., 2015Kean J. Rainey S.M. McFarlane M. Donald C.L. Schnettler E. Kohl A. Pondeville E. Fighting arbovirus transmission: natural and engineered control of vector competence in Aedes mosquitoes.Insects. 2015; 6: 236-278Crossref PubMed Scopus (51) Google Scholar), reducing viral load will directly result in a reduction of transmission rate. Natural selection will thus favor arbovirus mutants that can restore a higher transmission rate by evading or suppressing the resistance mechanism. It is worth noting that recent developments in genetic engineering strategies, such as simultaneously targeting multiple conserved regions of the viral genome, or different steps of the viral cycle, will minimize the probability of viral escape (Buchman et al., 2019Buchman A. Gamez S. Li M. Antoshechkin I. Li H.H. Wang H.W. Chen C.H. Klein M.J. Duchemin J.B. Paradkar P.N. et al.Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs.Proc. Natl. Acad. Sci. USA. 2019; 116: 3656-3661Crossref PubMed Scopus (57) Google Scholar). In the case of decreased mosquito tolerance, the transmission rate will remain unchanged because, by definition, the viral load is unchanged. However, the duration of transmission will decrease because infected vectors have a shortened lifespan. In that case, natural selection will favor arbovirus mutants that are less virulent (attenuated) in the vector to restore a longer duration of transmission. The extent to which the viral load will decrease depends on the new evolutionary optimum determined by the trade-off between virulence and transmission rate (Figure 3). We cannot ignore the possibility that arbovirus mutants with reduced virulence in the mosquito may have increased virulence in the vertebrate host. Future studies will be necessary to evaluate this possibility and determine whether reduced transmissibility could be offset by increased virulence in the vertebrate host. Note that our reasoning to compare the evolutionary response to tolerance-based and resistance-based strategies for disease control relies on at least three simplifying assumptions that condition our conclusions. First, we assume that manipulation of mosquito resistance and tolerance will not result in major fitness costs in the absence of the arbovirus. However, genetic manipulation of mosquitoes may result in constitutive fitness costs due to inbreeding and/or transgene expression (Moreira et al., 2004Moreira L.A. Wang J. Collins F.H. Jacobs-Lorena M. Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development.Genetics. 2004; 166: 1337-1341Crossref PubMed Scopus (108) Google Scholar). Second, we assume that the change in mosquito resistance or tolerance is specific to arboviral infections and does not influence other viral infections of mosquitoes, such as infection by insect-specific viruses. Wolbachia transfection in Ae. aegypti, for example, suppresses replication of some insect-specific viruses (Schnettler et al., 2016Schnettler E. Sreenu V.B. Mottram T. McFarlane M. Wolbachia restricts insect-specific Flavivirus infection in Aedes aegypti cells.J. Gen. Virol. 2016; 97: 3024-3029Crossref PubMed Scopus (40) Google Scholar) and possibly enhances that of others (Amuzu et al., 2018Amuzu H.E. Tsyganov K. Koh C. Herbert R.I. Powell D.R. McGraw E.A. Wolbachia enhances insect-specific Flavivirus infection in Aedes aegypti mosquitoes.Ecol. Evol. 2018; 8: 5441-5454Crossref PubMed Scopus (25) Google Scholar). It is likely that natural tolerance mechanisms act across a broad spectrum of viruses. Therefore, specificity against human pathogenic arboviruses could be achieved by complementing a general but subtle loss of tolerance with a more specific, artificial suicide-mediating mechanism. Third, we assume that we can manipulate resistance and tolerance independently from each other. Resistance and tolerance were found to be genetically correlated in a mouse model (Råberg et al., 2007Råberg L. Sim D. Read A.F. Disentangling genetic variation for resistance and tolerance to infectious diseases in animals.Science. 2007; 318: 812-814Crossref PubMed Scopus (534) Google Scholar), reflecting the existence of pleotropic genes that contribute to both phenotypes. The endoribonuclease Dicer-2, for example, is a cornerstone of mosquito resistance to arbovirus infection (Sánchez-Vargas et al., 2009Sánchez-Vargas I. Scott J.C. Poole-Smith B.K. Franz A.W. Barbosa-Solomieu V. Wilusz J. Olson K.E. Blair C.D. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.PLoS Pathog. 2009; 5: e1000299Crossref PubMed Scopus (335) Google Scholar), but it may also contribute to tolerance through the viral DNA biogenesis (Poirier et al., 2018Poirier E.Z. Goic B. Tomé-Poderti L. Frangeul L. Boussier J. Gausson V. Blanc H. Vallet T. Loyd H. Levi L.I. et al.Dicer-2-dependent generation of viral DNA from defective genomes of RNA viruses modulates antiviral immunity in insects.Cell Host Microbe. 2018; 23: 353-365Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The proof of principle that we can manipulate mosquito tolerance (Goic et al., 2016Goic B. Stapleford K.A. Frangeul L. Doucet A.J. Gausson V. Blanc H. Schemmel-Jofre N. Cristofari G. Lambrechts L. Vignuzzi M. et al.Virus-derived DNA drives mosquito vector tolerance to arboviral infection.Nat. Commun. 2016; 7: 12410Crossref PubMed Scopus (134) Google Scholar) supports the idea that reducing mosquito tolerance to arbovirus infection could be used as a disease control strategy (Figure 1). In this review, we have provided theoretical arguments supporting the idea that reducing tolerance should be more evolutionarily robust than increasing mosquito resistance to arbovirus infection. However, a number of scientific, logistical, and ethical challenges remain to be evaluated before converting this idea into a realistic and sustainable intervention for disease control. Additional knowledge is necessary to better understand the mechanisms of mosquito tolerance and their specificity. The strategy relies on the assumption that off-target effects are minimal and therefore loss of tolerance is an ecologically sound strategy. In other words, the loss of tolerance must be achieved specifically for arbovirus-infected mosquitoes, in the absence of fitness cost for arbovirus-free mosquitoes. Understanding the mechanisms of tolerance will be essential to design a method to safely and specifically manipulate it. Moreover, the mechanism employed to reduce mosquito tolerance will have to be delivered and remain effective under field conditions. Delivery may rely on mass releases of gene drive systems that are being developed for other candidate methods of vector population modification (Flores and O'Neill, 2018Flores H.A. O'Neill S.L. Controlling vector-borne diseases by releasing modified mosquitoes.Nat. Rev. Microbiol. 2018; 16: 508-518Crossref PubMed Scopus (163) Google Scholar). Like for other innovative vector control strategies under development, the proof of concept is only the very first step toward successful implementation (Achee et al., 2019Achee N.L. Grieco J.P. Vatandoost H. Seixas G. Pinto J. Ching-Ng L. Martins A.J. Juntarajumnong W. Corbel V. Gouagna C. et al.Alternative strategies for mosquito-borne arbovirus control.PLoS Negl. Trop. Dis. 2019; 13: e0006822PubMed Google Scholar). The public health value of an entomological intervention is ultimately established through large scale field trials with epidemiological endpoints. Such a difficult and time consuming process makes it crucial to preferentially invest in candidate disease control strategies that are evolutionarily sustainable. We thank Cassandra Koh, Sarah Merkling, and Lluis Quintana-Murci for insightful discussions and critical reading of the manuscript. This work was supported by the French Government's Investissement d'avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID) to L.L. and M.-C.S. L.L. is supported by Agence Nationale de la Recherche (grants ANR-16-CE35-0004-01 and ANR-17-ERC2-0016-01). M.-C.S. is supported by the European Research Council (FP7/2013-2019 ERC CoG 615220). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. M.-C.S. is funded by the DARPA PREEMPT program Cooperative Agreement D18AC00030. The content of the information does not necessarily reflect the position or the policy of the U.S. Government, and no official endorsement should be inferred.