Microbes and Infection 15 (2013) 755e758www.elsevier.com/locate/micinf

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Dope or die*

In 1818, the father of Dipterology, German entomologistJohann Wilhelm Meigen added some 19 lithographic platesabout non-european Diptera to his work “Die Fliegen” (theflies), containing several drawings of mosquitoes he obviouslydisliked as he named them Anopheles, literally “useless”, andAedes, meaning “odious” in Ancient Greek. The ones tostrongly disagree with Dr. Meigen are respectively the parasitesPlasmodium, the causative agents of malaria, and a range ofviruses, conveying charming diseases like dengue, Yellow andWest Nile fever, who make great use of these flying vectors toget ready to infect humans. Mankind’s trouble with malariaarose concomitantly with human sedentism; some records datefrom the 3000 BC in China, and dengue was first describedaround 300 BC. Nevertheless, major public concerns aroserather recently during the second half of the 20th century. Atfault is the arrival of once far-away tropical diseases in thedeveloped countries, due both to climate change and global-ization. The West Nile Virus, originating from Africa, made itsapparition in North America only in 1999 and keeps USauthorities still busy since the 2012 summer record temper-atures while, Aedes aegypti caused dengue outbreaks in Floridain 2009 and 2010, several cases in France in 2010 and invadespopular tourist destinations in Latin America and South EastAsia [2,3].

Wiping out the Corpus delicti, the parasites and viruses,sounds rather intuitive but is hampered by their high muta-genic and adaptive abilities; as a consequence, no approvedvaccines are available for dengue or malaria, and not for lackof trying [2,4]. That’s how the mosquitoes arrived in the sci-entist’s cross hairs, leading to the idea of exterminating all themosquitoes. Mosquitoes are not especially popular amonghumans, thus most of the latter were pretty fine with this. Noone was going to miss them anyway. Between 1950 and 1970,insecticides became very popular until it became clear thatthese expensive chemicals did not reach hidden larvae, pushedresistance and did no much good to humans either [2,5].Concomitantly with the necessity to review the strategies,

* A short history of genetically transformed mosquitoes and article highlight

of “Overexpression of phosphatase and tensin homolog improves fitness and

decreases Plasmodium falciparum development in Anopheles stephensi” by

Eric S. Hauck et al. [1].

1286-4579/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.micinf.2013.07.007

toying around with genes and genomes became commonpractice and the link was quickly established: releasinggenetically transformed mosquitoes in charge of either killingtheir own species in a suicide mission or replacing them by a2.0 version resistant to stowaways.

There are only a couple of problems. People are quitesusceptible when it comes to genetically modified organisms(GMOs). The right gene has to be chosen. The transgenicmosquitoes should be produced in huge quantities. Their fit-ness must not be impaired. And people are really susceptibleto GMOs and corn-eating rats.

Nevertheless, many international conferences were heldand much progress made during the last fifty decades, mainlyin the extermination strategy termed SIT for Sterile InsectTechnique. The principle consists in releasing huge quantitiesof insects, usually males, able to mate but not no produce aviable progeny. Credits for the idea go to American ento-mologists Knipling and Bushland in the 1950s who proposedto release X-rays sterilized males. Ideally, they outnumbercompetent males and copulate with wild females withoutfertilizing their eggs. In many insect species, females mateonly once. The technique proved successful for eradication ofthe screwworm fly in North and Central America and is usedsince decades to suppress the Mediterranean fruit fly [3,6,7].However, several trials on mosquitoes in the 70s and 80sshowed that the latter are too sensitive to X-ray or UV-inducedsterilization and the males are not able to mate efficiently[2,3].

Another sterilization strategy was needed and the step ofactive meddling with the genome taken. The one with thehighest media coverage is the RIDL e release of insects with adominant lethal gene e approach. The famous lethal gene israther an incomplete Tet-Off system: the tTA coding gene anda fluorescent gene are injected into mosquito larvae and thetTA protein produced via activation of its own promoter, butno other tTA binding promoter is present in the larval genome.It turns out that tTA protein production kills larvae and pupaebut not the adult for some unclear reasons, maybe proteinoverload or transcription factor monopolization [4]. tTAexpression in the laboratory is repressed by the antibiotictetracycline until the achievement of production of highquantities of transgenic animals and pupae size-basedmechanical selection of males. Released males are meant tomate with females and pass on the tTA encoding gene to their

sson SAS. All rights reserved.

756 Highlight / Microbes and Infection 15 (2013) 755e758

progeny which will die at early stages lacking tetracycline [6].Two small-scale field experiments were conducted in 2009 and2010, one on the Grand Cayman island in the Caribbean andone in an inhabited forest of Pahang in Malaysia, providing theproof of principle that the millions of released RIDL malescould mate with wild females, even if their mating success wasreduced by 50%, had a comparable lifespan and lower dis-persal than wild males and could locally reduce the number ofmosquitoes [2,3,6]. Needless to say that criticism regardingsmall sampling scale, collection randomness and equalresponsiveness of RIDL and wild males to traps was imme-diate, moreover the fact that a British company, Oxitec, put itshands on the technique fueled aggressive comments of inter-nauts. Nevertheless, SIT strategies eradicated Culex quinque-fasciatus from Myanmar [7]. Other promising techniques arebased on Wolbachia infected vectors, concurrencing denguevirus and RNA interference [8].

What are the criteria of the acceptable transgenic sterilemosquito [7]? A major problem is the fitness reduction oftransgenic males due to either the transgene or the muta-genesis. However, laboratory conditions poorly mimic thewild, wild world [5,6]. Computational models predict that amating fraction of 13e57% and a heterozygous populationwith higher fitness than the wild-type are required for sup-pression [2,9]. The less efficient the mating of transgenicmales with wild females, the more animals have to be releasedrepetitively until complete extermination [7]. This leads to thenext issue: the financial requirements and logistics for large-scale insect production, rearing, strategy planning, transport,quality control, release and monitoring, which are not withinreach of most developing countries. Things also becomelegally problematic when one country wants to use transgenicanimals whereas its neighbour does not, while insects knowlittle about frontiers [2].

The most delicate issue is obviously the transgene itself.Alteration of transgenic effectors through the instability anddegeneration of transposable elements, used to insert thetransgene like the piggyback system for RIDL, or horizontaltransmission could cause unanticipated effects which willprobably not create a flesh-eating killer mosquito but wouldrather annihilate the project’s goal, favour resistance emer-gence or threaten the local ecosystem [7]. It has been proventhat re-mobilizing non-autonomous transposons is very diffi-cult even in transposase presence [4] but very difficult is notsynonymous to impossible and if we have to keep one thing inmind, it is that even ridiculously small probabilities e like0.1% of viable progeny for example e end up to make a lotwhen billions of repeatedly released mosquitoes are on theagenda. No genotype present in the wild population, or anynatural condition, should also be possible to complement thetransgenic trait. Yet, tetracycline is used as an antibiotic in thenatural environment (agriculture, water tanks). Insertion itselfalready can have unexpected effects due to a position effect.Insertion of EGFP into Anopheles gambiae, meant to be femalespecific, turned on in both sexes and even more in males,possibly because of cryptic promoters [4]. Targeted insertionand whole genome sequencing are mandatory. However,

exterminating an entire species leaves behind an empty eco-logical niche which could be filled by many other nasty beings.

Many scientists propose enthusiastically alternative meth-ods like the team of Shirley Luckhart proposing to dopeAnopheles stephensi by PTEN phosphatase overexpression fora better intestinal health, unable to support pathogen develop-ment [1]. Other possible candidates are naturally occurring orsynthetic antimicrobial peptides or antibodies against mosquitomidgut proteins [5]. So why does research stick mainly to thekill-them-all strategy? Because it has an emergency brake. SITcan be reversed by simply stopping the releases when things donot go as expected, fertile transgenic mosquitoes can’t [7]. Andwon’t making fitter mosquitoes lead to making fitter parasites?Population genetics may be supported by unlimited complexmodels, but the final answer is only given by trying, watchingand coming up with explanations afterwards.

Not to forget about politics sometimes getting in the way.The joint World Health Organization (WHO) and IndianCouncil of Medical Research (ICMR) efforts, financed parti-ally by the US, planning to release sterile males against C.quinquefasciatus, A. stephensi and A. aegypti near New Delhicame to an abrupt end in the mid 70s when some Indian mediaand politicians accused the project to be a cover for biologicalwarfare purposes because of some American experimentsinvolving mosquitoes, yellow fever and the inhabited Bakerislands [7,10]. Forty years later, over a million cases of malariaare counted in India each year. In El Salvador, promising pilotelimination trials of Anopheles albimanus ended in the civilwar which started in 1980 [7].

Finding solutions becomes urgent. If no additional meas-ures are taken, malaria incidence will have doubled in 20years. The latest progress of techniques and technology,combining new insights in genetics and epigenetics, wholegenome sequencing and targeted mutagenesis as well asrefined production and monitoring possibilities argue for giv-ing the transgenic mosquitoes a try, as part of a wider programof pest and public opinion managing [4].

By the way, did you notice that mosquitoes are alongsidewith ships among the rare non-human english nouns to beaddressed as “she”?

Biosketch e Shirley LuckhartDr. Shirley Luckhart completed her Bachelor of Science in

1986 from the University of Florida in Natural ResourceConservation, a Master of Science in 1990 in Entomologyfrom Auburn University, and a PhD in Entomology fromRutgers University in 1995. She was a National ResearchCouncil Fellow at Walter Reed Army Institute of Researchfrom 1995 to 1998 and an adjunct faculty member in theDepartment of Preventative Medicine and Biometrics at theUniformed Services University of the Health Sciences. Shethen joined the faculty in the Department of Biochemistry atVirginia Tech in 1998 and remained at VT until 2004. In 2004,she moved to UC Davis, where she is now Professor ofMedical Microbiology and Immunology in the School ofMedicine. Dr. Luckhart’s research has focused extensively onthe ecology, molecular cell biology, and biochemistry ofhostepathogen interactions in vector-borne diseases, including

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filariasis, onchocerciasis, Lyme disease, and malaria. Hercurrent research is focused on malaria, specifically on innateimmune mechanisms that influence parasite transmission. Shehas collaborations in Colombia, Mali, Kenya, and Cameroon.

Interview with Shirley Luckhart

What triggered your interest in the link between PTEN andPlasmodium falciparum?

Wehavebeenworkingwith collaboratorseDr.MichaelRiehleof theUniversity ofArizona andDr.MarkBrown of theUniversityof Georgiae on insulin/insulin-like growth factor signalling (IIS)in the mosquito A. stephensi for the past 6 years. Our work hasclearly demonstrated that IIS controls malaria parasite infection inA. stephensi. Our work on PTEN, an inhibitor of IIS, was a naturalextension of our previous work on Akt, an activator of IIS.

What was your first reaction when you faced the results?Did you expect them?

We predicted, based on extensive studies in Caenorhabditiselegans and Drosophila melanogaster, that Akt overexpressionin the mosquito midgut should decrease mosquito lifespan andincrease susceptibility to malaria parasite infection. While wesaw an Akt-dependent reduction in lifespan, we also observedvery high resistance to parasite infection (Corby-Harris, et al.,PLoS Pathog. 6 (7) (2010) e1001003; Luckhart, et al., PLoSPathog. 9(2) (2013) e1003180). In fact, this work was widelyaddressed in the popular press as the first development of“malaria-proof mosquitoes” and named by Time Magazine oneof the top medical inventions for 2010. While this was cer-tainly unexpected, we did continue to believe that PTENoverexpression should generate phenotypes that were con-sistent with those reported for IIS inhibition in model inver-tebrates. These predictions held true: PTEN overexpressionresulted in increased lifespan and increased resistance tomalaria parasite infection. Hence, we now have a strategy to

begin to work toward the development of genetically modifiedmosquitoes that are resistant to parasite infection, but that alsolive longer than wild type mosquitoes.

How will the project go on?

Based on our 2013 paper in PLoS Pathogens, we areexcited to move forward to understand how IIS-dependentmitochondrial dynamics controls important phenotypes oflifespan and parasite infection. Indeed, cutting-edge studies inmammalian biology have now confirmed that “mitochondrialhealth” functions as an essential check-point for an incrediblybroad array of physiologies, so this is likely true in mosquitoesas well. It will be fascinating to see early origins of mito-chondrial control of complex physiologies.

What is the take-home message of the article?

Manipulation of IIS can lead to enhanced resistance toPlasmodium falciparum infection and extended lifespan, aperfect platform from which to develop resistant mosquitoesthat are competitive with wild mosquitoes under field con-ditions as a novel strategy for malaria control.

Do you have a personal motto, quote or leading sentence?

Have plan B, C, D, E, F.. ready when A doesn’t work.What advice would you give to the young next-generation

scientists?

This is a tough business, but it is incredibly rewarding ifyou can develop the siege mentality to succeed.

What is your favourite hang-out method after a tough dayat the lab?

Have a nice glass of wine and cook dinner with myhusband e he’s the chef, I’m the sous chef.

� Mosquitoes overexpressing PTEN displayed areduced prevalence and intensity of P. falciparuminfection

� No significant difference in the expression ofimmunity-related genes was observed betweenPTEN overexpressing A. stephensi and control

� Markers of stem cell development and autophagywere increased in the midgut of mosquitoes over-expressing PTEN compared to controls, in parallelwith enhanced epithelial impermeability to beads

758 Highlight / Microbes and Infection 15 (2013) 755e758

In your opinion, what are the three most important (sci-entific) discoveries of the last decade?

Patients have been identified who can clear HIV infection.Stem cells can be programmed to generate tissues. The studyof invertebrates as a foundation for our understanding ofhuman immunology can win the Nobel Prize in Physiology orMedicine.

If you could travel back in time e what historical person-ality would you like to meet and what scientific discovery toassist to?

I would like to go back to work with Louis Pasteur when heestablished the germ theory of disease through his work tosave the silk industry in France. Pasteur recognized that seg-regating sick silkworms from healthy-appearing silkwormsprevented the spread of disease by eliminating the causativemicrobes. Hence, the basis of our understanding of infectiousdiseases has critical foundations in the study of insects. Iwould like to tell Pasteur that in addition to this fantastic workthat he needed to promote the study of insects as windows toevolutionarily conserved phenomena in mammals. He wasobviously a man ahead of his time.

If you could travel forth in time e what eventual inventionwould you like to check out?

Simple: the group of tools/ideas that were used to eradicatemalaria from the planet.

Background

� Dengue fever, caused why a virus conveyed byAedes mosquitoes, affects 50e100 million indi-viduals per year. The incidence increased 30 foldover the last 50 years. Haemorrhagic dengue causes500 000 cases per year and kills 25 000 persons

� There are over 200 million malaria cases and over600 000 deaths caused by malaria per year world-wide. The causative agent is parasites namedPlasmodium mostly transmitted to human bymosquitoes of the Anopheles family

� A. stephensi is the main mosquito vector of malariacaused by Plasmodium falciparum in Indian urbanareas where it breeds in water-bodies, artificialcontainers, building walls and water tanks

� Trials with sterile or transgenetic mosquitoes havebeen undertaken since 1960, mainly at small scalefor experimental purposes

In A Nutshell

� PTEN overexpression A. stephensi midgut inhibitedbasal and insulin-induced FOXO phosphorylation,a downstream effector of IIS, and increased themosquitoes’ lifespan

Acknowledgements

Acknowledgements to A. Ludl for providing me with lit-erature and T. Talvard for text correction.

References

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Sophia HafnerUniv. Paris Diderot, Epigenetics and Cell Fate, UMR 7216

CNRS, Sorbonne Paris Cite, 75013 Paris, FranceE-mail address: sophia.hafner@univ-paris-diderot.fr

20 July 2013