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Malaria Control with Genetically Manipulated Insect Vectors

November 2002
Science 298(5591):119-21

DOI:10.1126/science.1078278

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PubMed

Authors:

Luke Alphey

The Pirbright Institute

Charles Ben Beard

Centers for Disease Control and Prevention

Peter Billingsley

Sanaria Inc.

Maureen Coetzee

University of the Witwatersrand

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At a recent workshop, experts discussed the benefits, risks, and research priorities associated with using genetically manipulated insects in the control of vector-borne diseases.

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DOI: 10.1126/science.1078278

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Malaria Control with Genetically Manipulated Insect

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and effectively transmit dengue virus even at

very low population densities because they pref-

erentially and frequently bite humans (23). A

successful GMM dengue control program that

falls short of vector eradication will result in a

reduction in human herd immunity and a corre-

sponding decrease in already low transmission

threshold levels. Because there is no commercial-

ly available vaccine or clinical cure for dengue,

predicting and testing transmission thresholds is

among the most important unanswered questions

in dengue epidemiology and GMM-based control

approaches.

Quantitative Analyses of Mosquito

Biology, Disease, and Control by GMM

A goal of future quantitative analyses should be

to accurately predict outcomes of proposed inter-

ventions instead of simulating events retrospec-

tively. For example, continental-scale predictions

of malaria disease burden are currently being

made on the basis of remotely sensed environ-

mental data that influence mosquito population

dynamics and, in turn, patterns of pathogen trans-

mission (24). Simulation models have been used

to predict entomological thresholds for dengue

transmission (25). Mathematical models have

been developed to identify parameters required to

predict the dynamics of transgene drive mecha-

nisms in vector populations (5, 6, 13, 26). Dif-

ferent drive strategies have been examined and

predictions made for the likely success of each

(5). An analysis of population genetics and epi-

demiology has concluded that in areas of intense

malaria transmission, GMM control programs

will have little if any effect unless mosquito

refractoriness is very close to 100% (13).

Conclusions

The meeting participants reached consensus on

four procedural issues. First, there is an urgent

need to develop uniform processes for dealing

with the ethical, legal, and social issues related to

GMM technology (27). It would be most helpful

if an international body like the World Health

Organization established guidelines, regulatory

mechanisms, and safety, containment, and con-

servation protocols. Second, for the GMM ap-

proach to be initially successful and ultimately

sustainable, its proponents must identify and de-

velop the capacity for human resources and re-

search infrastructure at sites earmarked for tech-

nology evaluation and long-term application.

Third, continued evaluation of GMM technology

will require semi-field facilities (such as large

outdoor cages), followed by release of GMM on

isolated oceanic or ecological islands that have

been thoroughly characterized with respect to the

genetic and ecological makeup of local mosquito

vector populations and site-specific patterns of

pathogen transmission and disease. Fourth, in

addition to population replacement, genetic strat-

egies for mosquito population reduction [such as

RIDL (release of insects carrying a dominant

lethal) and negative heterosis] in isolated urban

areas merit consideration (28).

Addressing these goals will require coordi-

nated interaction among scientists from diverse

disciplines. Only by studying the system in total

will we gain greater insight into the complexity

of interactions that are essential for the design,

implementation, and evaluation of progressively

more successful disease management strategies.

Such an ambitious agenda will require adequate

funding, collaboration between ecologists and

molecular geneticists, recruitment of expertise

from outside the vector-borne disease arena,

training for young scientists, and the expectation

of a sustained effort. The longitudinal field stud-

ies required to address some of the ecological

issues identified will last a decade or more. In all

these actions, people from the countries where

GMM technology is most likely to be applied

need to be more fully involved.

References and Notes

1. J. Trape, G. Pison, A. Spiegel, C. Enel, C. Rogier, Trends

Parasitol. 18, 224 (2002).

2. W. Takken, Trop. Med. Int. Health, in press.

3. A. A. James, in Insect Transgenesis: Methods and

Applications, A. M. Handler, A. A. James, Eds. (CRC

Press, Boca Raton, FL, 2000), pp. 319–333.

4. M. Enserink, Science 297, 30 (2002).

5. M. Turelli, A. A. Hoffmann, Insect Mol. Biol. 8, 243

(1999).

6. J. M. Ribeiro, M. G. Kidwell, J. Med. Entomol. 31,10

(1994).

7. F. M. Okanda et al., Malaria J. 1, 10 (2002).

8. M. J. Donnelly, F. Simard, T. Lehmann, Trends Parasi-

tol. 18, 75 (2002).

9. A. della Torre et al., Science 298, 115 (2002).

10. Y. T. Toure´ et al., Med. Vet. Entomol. 12, 74 (1998).

11. C. E. Taylor, Y. T. Toure´, M. Coluzzi, V. Petrarca, Med.

Vet. Entomol. 7, 351 (1993).

12. N. Lorimer, L. P. Lounibos, J. L. Petersen, J. Econ.

Entomol. 69, 405 (1976).

13. C. Boe¨te, J. C. Koella, Malaria J. 1, 3 (2002).

14. J. Ito, A. Ghosh, L. A. Moreira, E. A. Wimmer, M.

Jacobs-Lorena, Nature 417, 452 (2002).

15. C. Boe¨te, J. C. Koella, Trends Parasitol., in press.

16. N. J. White et al., Lancet 353, 1965 (1999).

17. I. S. Novella et al., J. Mol. Biol. 287, 459 (1999).

18. C. Boe¨te, R. E. L. Paul, J. C. Koella, Parasitology 125,93

(2002).

19. J. D. Charlwood et al., Am. J. Trop. Med. Hyg. 59, 243

(1998).

20. T. A. Smith, R. Leuenberger, C. Lengeler, Trends Para-

sitol. 17, 145 (2001).

21. C. A. Maxwell et al., Trop. Med. Int. Health, in press.

22. A. C. Morrison, A. Getis, T. W. Scott, unpublished

data.

23. T. W. Scott et al., J. Med. Entomol. 37, 89 (2000).

24. D. Rogers, S. E. Randolph, R. W. Snow, S. I. Hay,

Nature 415, 702 (2002).

25. D. A. Focks, R. J. Brenner, J. Hayes, E. Daniels, Am. J.

Trop. Med. Hyg. 62, 11 (2000).

26. A. E. Kiszewski, A. Spielman, J. Med. Entomol. 35, 584

(2002).

27. L. Alphey et al., Science 298, 119 (2002).

28. L. Alphey, M. Andreasen, Mol. Biochem. Parasitol.

121, 173 (2002).

29. We thank the following Wageningin meeting partic-

ipants for helpful comments: K. Aultman, P. Billings-

ley, D. Charlwood, C. Curtis, J. Edman, J. Koella, G.

Lanzaro, S. Lindsay, P. Lounibos, D. O’Brochta, S.

Randolph, W. Reisen, D. Rogers, M. Sabelis, A. Spiel-

man, C. Taylor, and Y. Toure´.

VIEWPOINT

Malaria Control with Genetically

Manipulated Insect Vectors

Luke Alphey,

C. Ben Beard,

Peter Billingsley,

Maureen Coetzee,

Andrea Crisanti,

Chris Curtis,

Paul Eggleston,

Charles Godfray,

Janet Hemingway,

Marcelo Jacobs-Lorena,

Anthony A. James,

Fotis C. Kafatos,

Louis G. Mukwaya,

Michael Paton,

Jeffrey R. Powell,

William Schneider,

Thomas W. Scott,

Barbara Sina,

Robert Sinden,

Steven Sinkins,

Andrew Spielman,

Yeya Toure´,

Frank H. Collins

At a recent workshop, experts discussed the beneﬁts, risks, and research

priorities associated with using genetically manipulated insects in the

control of vector-borne diseases.

This is a partial report of a workshop—Genet-

ically Engineered Arthropod Vectors of Human

Infectious Diseases—jointly sponsored by the

World Health Organization, the MacArthur

Foundation, the National Institute of Allergy

and Infectious Diseases, and London’s Imperial

College—originally planned for 12 September

2001 in London (but reconvened in successive

sessions later in London and Atlanta). These

workshops sought to encourage communication

between the laboratory-oriented molecular bi-

ologists, whose work had suggested the poten-

tial of genetic control strategies, and the popu-

lation geneticists, ecologists, and public health

specialists, whose involvement would be cru-

cial in moving the work beyond the laboratory.

The meeting participants were charged with

considering the benefits and risks of using ge-

netically engineered arthropod vectors as public

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health tools and mapping out a research agenda

for their development. The task of engineering

different vector species and the risks associated

with various methods of genetic engineering

are vastly different and could not be addressed

in a single report. What follows is the consen-

sus of the working group on germline-trans-

formed organisms developed for control of ma-

laria transmission (authors listed above) and

other participants. The reports of the working

groups on paratransgenesis (transformation of

obligate symbionts in insects) and on other

vector-borne diseases will be presented in the

near future.

In 1991, a scientific workshop in Arizona

assessed the prospect for malaria control by

genetic manipulation of vector populations (see

the Viewpoint by Morel et al. on page 79) (1).

The basic concept of genetic control of vector-

borne diseases was proposed by Curtis in 1968

(2), but major advances in the molecular ma-

nipulation of Drosophila melanogaster during

the 1980s encouraged reevaluation of this idea.

The WHO/TDR summary document of the

meeting laid out a clear list of research aims that

would have to be met before a genetic control

strategy could be field tested (3). These aims

fell into three categories: (i) the development of

genetic engineering tools that could be used

with malaria vectors; (ii) the identification of

effector genes that could block parasite trans-

mission; and (iii) the development of effective

methods for driving these effector genes to

fixation in natural vector populations.

The first two aims have been largely

achieved. Several different but effective

methods of germline transformation have

been developed and used in at least three

species of malaria mosquito vector (4– 6);

two different laboratories have developed ge-

netic constructs that significantly reduce vec-

tor competence in experimental malaria mod-

els (7, 8). A large set of molecular markers

has been developed and is being used in

studies of gene flow and population structure

in anopheline malaria vectors (9–13). But

there has been no significant progress in de-

veloping methods for driving desirable genes

into wild populations and especially for en-

suring the necessary unbreakable linkage be-

tween the drive system and the gene to be

driven (see the Viewpoint by Scott et al.on

page 117) (14).

Consideration of the potential use of ge-

netically modified organisms (GMOs) is

driven by the realization of the enormous

human cost of diseases like malaria, and of

the inadequacy of present control measures.

Perhaps the most important theme emerging

from the workshop was the recognition that

control strategies involving GMOs could po-

tentially provoke serious public mistrust and

resistance to their implementation. Therefore

it was strongly recommended that all work

leading to the development of specific genet-

ic control strategies targeted at malaria vec-

tors should involve both public health spe-

cialists and scientists from disease-endemic

countries and (where possible) the general

public in areas where field trials could be

implemented. Because field trials of geneti-

cally modified mosquitoes would have to be

preceded by long-term, longitudinal studies

of potential field-trial sites, the local commu-

nity and its own scientists and health experts

can easily be involved.

The goal of producing GMOs intended to

benefit human health has been perceived more

favorably by the public than that of producing

GMOs for agricultural or domestic animal re-

search. However, meeting participants strongly

argued that this positive public perception could

be rapidly undermined by an actual field trial of

a transgenic arthropod that failed to provide a

significant and tangible health benefit to the

resident human community. It was therefore

recommended that all preliminary research de-

signed to lead to field trials of the efficacy of a

transgenic arthropod-based disease control

strategy should involve fully contained labora-

tory or cage environments. Release should be

permitted only when all relevant parameters

had been investigated in either contained envi-

ronments or in open field studies that did not

involve transgenic arthropods. Furthermore,

field trials involving release of transgenic ar-

thropods should take place only when all mem-

bers of both scientific and local community

review groups were assured that such trials had

a very high probability of producing a signifi-

cant and measurable public health benefit for

the local community.

Many important ecological and popula-

tion genetic issues must be understood before

any release program can be contemplated,

and such issues will be specific not only to

individual vector species but also to local

populations (see the Viewpoint by Scott et al.

on page 117) (14). Understanding the dynam-

ics of a natural population will require years

of study, with the time frame dependent on

the stability and repeatability of yearly cy-

cles. Thus, given progress in the laboratory, it

is important to start the ecological and pop-

ulation genetic study of potential target pop-

ulations soon, as this will be the biggest

scientific limitation to implementing genetic

control field trials. A large number of tech-

nical problems will have to be addressed,

ranging from the feasibility of producing an

effective release strain to the design and as-

sessment of release strategies with specifical-

ly predicted goals. To address such problems

will require the involvement of ecologists and

population geneticists. Most participants rec-

ommended that study of potential field-trial

sites should be initiated immediately at mul-

tiple different locations, recognizing that the

initial phase of fieldwork might show one or

more of the selected sites to be unsuitable.

Because the biology of vector populations at

any such site would have to be studied for

many years before field trials could be de-

signed, the community cannot investigate dif-

ferent sites sequentially.

GMOs could be used in either of two ways

for malaria control. The initial concept (ex-

pressed in the 1991 meeting) was to engineer

mosquitoes with an altered phenotype that

would be introduced into the population in such

a way that the new trait would spread and

become dominant. These strategies target the

malaria parasite, rather than the mosquito itself,

for reduction. There is an immediate research

need for the study of drive systems in Anopheles

species. These drive systems also present a po-

tential hazard because they may generate unin-

tended phenotypes and have unforeseen, poten-

tially harmful ecological effects. Autonomous

transposons, for example, could increase the

mutation rate through multiple genomic inser-

tions, leading to unanticipated alterations in the

biology of the target species. Tight linkage of

the drive system and the engineered gene is also

an important issue in that its loss in the progeny

of released mosquitoes could lead to loss of

public health efficacy and loss of the molecular

tool for future engineering efforts. Although

transposon and symbiont systems have garnered

the most attention to date, participants recog-

nized the need to explore any possible drive

system that could continue to propagate a re-

leased genetic construct through the target pop-

ulation after initial release.

An alternative use of genetic engineering for

malaria control takes a more traditional ap-

proach. This involves targeting the mosqui-

to population per se for reduction. Proposed

improvements in sterile insect techniques,

including release of insects carrying domi-

nant lethals (RIDL) (15), and other mech-

anisms of genetic sexing may alter the

prognosis for these strategies. In these sit-

uations the release of large numbers of

insects presents other specific challenges:

for example, the need to release only male

mosquitoes so as not to increase the number

or nature of mosquito bites per person per

night. In the absence of an existing drive

Oxford University, UK.

National Center for Infec-

tious Diseases, Centers for Disease Control and Pre-

vention, USA.

University of Aberdeen, UK.

South

African Institute for Medical Research, South Africa.

Imperial College, London, UK.

London School of

Tropical Medicine and Hygiene, UK.

Keele University,

UK.

Liverpool School of Tropical Medicine, UK.

Case

Western Reserve University, USA.

University of Cal-

ifornia, Irvine, USA.

European Molecular Biology

Laboratory, Germany.

Uganda Virus Research Insti-

tute, Uganda.

Health and Safety Executive, HSC, UK.

Yale University, USA.

Environmental Protection

Agency, USA.

University of California, Davis, USA.

Fogarty International Center, NIH, USA.

Harvard

School of Public Health, USA.

Special Programme

for Research and Training in Tropical Diseases (TDR),

WHO.

University of Notre Dame, USA.

4 OCTOBER 2002 VOL 298 SCIENCE www.sciencemag.org120

T HE M OSQUITO G ENOME: A NOPHELES GAMBIAE

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system, participants considered the use of

inundative release of refractory mosquitoes

as a strategy for limited field-testing of the

performance of specific genetically engi-

neered vector strains. Although considered

suitable only for a small vector population

with limited interpopulation gene flow

(such as a real or ecological island setting),

the ability to limit or quickly control un-

foreseen risks in the genetic manipulation

of an island population will be important in

early-stage trials designed to demonstrate

the efficacy of particular genetic modifica-

tions of the vector population.

Although there was support for continued,

intensive research in this area, a clear recom-

mendation emerged that there should be no pre-

cipitous releases of transgenic arthropods. The

malaria group was willing to recommend bar-

ring field trials of transgenic insects that were

designed solely for research; others felt that

initial field safety testing of the various individ-

ual elements of the engineered organism was

crucial to development. The parallel processes

of drug and vaccine development illustrate these

two views. For either product, and indeed for

engineered Anopheles mosquitoes, there is a

requirement for preliminary studies of safety

and efficacy in culture and in animal models

before the first clinical trial is initiated. With

many new drugs (other than cancer drugs), the

first human trials are performed in small num-

bers of normal healthy volunteers, and safety is

the end point examined. In these situations it

would be inappropriate to endanger patients

who are already sick by exposing them to a drug

candidate of unknown toxicity. By contrast,

when new vaccines are developed, they are most

often combined with adjuvants that improve

their potency or direct their effects to one or

more segments of the human immune system.

Under its current guidelines the U.S. Food and

Drug Administration does not allow investiga-

tion of the adjuvants alone without the vaccine

candidate being tested at the same time. The

malaria working group requires tangible bene-

fits at each phase of field testing. The other

working groups—discussing symbionts, trans-

ducing viruses, and other mechanisms of driving

traits into populations—decided to follow drug-

development protocols. These differences may

be appropriate given the different nature of the

engineering tools and the different risks associ-

ated with each one.

Despite nearly universal recognition that

enormous technical and sociological problems

must be overcome before the implementation of

genetic control strategies for malaria can be field

tested, participants concluded that public health

strategies incorporating transgenic vectors offer

the potential of health benefits. Participants

from disease-endemic areas, many of whom had

limited prior exposure to transgenic arthropod

research or policy discussions, were among the

most supportive and optimistic about the public

health goals such strategies hope to achieve.

Participants also noted that the broad scope of

biological research required for the development

of genetic control strategies is likely to contrib-

ute both to the more efficient application of

currently available control tools and to the de-

velopment of new approaches.

References

1. C. M. Morel, Y. T. Toure´, B. Dobrokhotov, A. M. J.

Oduola, Science 298, 79 (2002).

2. C. F. Curtis, Nature 218, 368 (1968).

3. Prospects for malaria control by genetic manipulation

of its vectors (TDR/BCV/MAL-ENT/91.3) (World

Health Organization, Geneva, 1991).

4. C. J. Coates, N. Jasinskiene, L. Miyashiro, A. A. James,,

Proc. Natl. Acad. Sci. U.S.A. 95, 3748 (1998).)

5. F. Caterrucia et al., Nature 405, 959 (2000).

6. G. L. Grossman et al., Insect Mol. Biol. 10, 597 (2001).

7. M. de Lara Capurro, Am. J. Trop. Med. Hyg. 62, 427

(2000).

8. J. Ito et al., Nature 417, 452 (2002).

9. D. Y. Onyabe, J. E. Conn, Heredity 87, 647 (2001).

10. M. J. Donnelly, M. C. Licht, T. Lehmann, Mol. Biol.

Evol. 18, 1353 (2001).

11. W. C. Black IV, G. C. Lanzaro, Insect Mol. Biol. 10,3

(2001).

12. C. Walton et al., Mol. Ecol. 10, 569 (2001).

13. R. Wang, F. C. Kafatos, L. Zheng, Parasitol. Today 15,

33 (1999).

14. T. W. Scott, W. Takken, B. G. J. Knols, C. Boe¨te,

Science 298, 117 (2002).

15. D. D. Thomas, C. A. Donnelly, R. J. Wood, L. S. Alphey,

Science 287, 2474 (2000).

VIEWPOINT

Malaria—a Shadow over Africa

Louis H. Miller

and Brian Greenwood

Reduction in severe disease and death from falciparum malaria in Africa

requires new, more effective and inexpensive public health measures. The

completed genomes of Plasmodium falciparum and its vector Anopheles

gambiae represent a big step toward the discovery of these needed tools.

The current focus of malaria control programs in

Africa is rightly on the management of sick

children through early treatment with effective

antimalarial drugs. However, this cannot be the

final strategy. The two first-line drugs, chloro-

quine and sulfadoxine/pyrimethamine (Fan-

sidar), are no longer effective in many parts of

East Africa where chloroquine resistance (intro-

duced from Asia) is rampant. Combinations of

new drugs may help to slow the emergence and

spread of resistant parasites (1), but control strat-

egies based on early treatment mean a never-

ending struggle to develop and deploy new drugs

before the Plasmodium malaria parasites become

resistant to existing drugs. Thus, the long-term

control strategy must be to interrupt the trans-

mission of this parasite. Unfortunately, this will

be extremely difficult in parts of Africa where

people may be bitten as many as 1000 times a

year by infected mosquitoes. Insecticide-treated

bed nets—now being vigorously promoted in

many parts of Africa—reduce bites from infect-

ed mosquitoes by as much as 90% (2). However,

their effectiveness is already under threat as a

result of the emergence of pyrethroid resistance

in Anopheles funestus in Mozambique and in A.

gambiae in agricultural areas of West Africa (3).

Household spraying with residual insecticides is

highly effective in reducing malaria in some

parts of Africa, but it is logistically demand-

ing, costly, and may have adverse environ-

mental effects.

There are many ways to reduce malaria

transmission, but none can provide a complete

block in transmission, particularly in the highly

endemic areas of Africa (4), and new approaches

are desperately needed (5). Publication of the

Plasmodium falciparum (6) and Anopheles gam-

biae genomes (7) represents a big step forward

in our search for new tools for controlling ma-

laria. Combined deployment of three strategies

that each have the potential to reduce malaria

transmission by 90%— drug treatment, vaccina-

tion, and vector control—should be sufficient to

stop transmission, even in highly endemic areas

of Africa. We will need to first test such strate-

gies in areas with a low intensity of transmission

before attempting the challenging task of pre-

venting malaria transmission in the highly en-

demic areas of Africa.

Anyone who has thought deeply about the

problem of reducing severe disease and death

from malaria in Africa realizes the crucial need

for a malaria vaccine. Pre-erythrocytic, blood-

stage, and transmission-blocking vaccines have

recently been developed by a number of groups

(8). Each type of vaccine has a part to play in

the complex, highly diverse epidemiology of

malaria and the associated variety of patterns of

Laboratory of Malaria and Vector Research, National

Institute of Allergy and Infectious Diseases, Bethesda,

MD 20892, USA.

Department of Infectious and Trop-

ical Diseases, London School of Hygiene and Tropical

Medicine, London WC1B 3DP, UK.

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Sep 2022
MALARIA J

Background South Africa has set a mandate to eliminate local malaria transmission by 2023. In pursuit of this objective a Sterile Insect Technique programme targeting the main vector Anopheles arabiensis is currently under development. Significant progress has been made towards operationalizing the technology. However, one of the main limitations being faced is the absence of an efficient genetic sexing system. This study is an assessment of an An. arabiensis (AY-2) strain carrying the full Y chromosome from Anopheles gambiae , including a transgenic red fluorescent marker, being introgressed into a South African genetic background as a potential tool for a reliable sexing system. Methods Adult, virgin males from the An. arabiensis AY-2 strain were outcrossed to virgin females from the South African, Kwazulu-Natal An. arabiensis (KWAG strain) over three generations. Anopheles arabiensis AY-2 fluorescent males were sorted as first instar larvae (L1) using the Complex Object Parametric Analyzer and Sorter (COPAS) and later screened as pupae to verify the sex. Life history traits of the novel hybrid KWAG-AY2 strain were compared to the original fluorescent AY-2 strain, the South African wild-type KWAG strain and a standard laboratory An. arabiensis (Dongola reference strain). Results The genetic stability of the sex-linked fluorescent marker and the integrity and high level of sexing efficiency of the system were confirmed. No recombination events in respect to the fluorescent marker were detected over three rounds of introgression crosses. KWAG-AY2 had higher hatch rates and survival of L1 to pupae and L1 to adult than the founding strains. AY-2 showed faster development time of immature stages and larger adult body size, but lower larval survival rates. Adult KWAG males had significantly higher survival rates. There was no significant difference between the strains in fecundity and proportion of males. KWAG-AY2 males performed better than reference strains in flight ability tests. Conclusion The life history traits of KWAG-AY2, its rearing efficiency under laboratory conditions, the preservation of the sex-linked fluorescence and perfect sexing efficiency after three rounds of introgression crosses, indicate that it has potential for mass rearing. The potential risks and benefits associated to the use of this strain within the Sterile Insect Technique programme in South Africa are discussed.

The effect of silencing immunity related genes on longevity in the naturally occurring Anopheles arabiensis mosquito population of Southwest Ethiopia

Thesis

Jan 2022

Serkadis Debalke Alene

Preparing an Insectary in Burkina Faso to Support Research in Genetic Technologies for Malaria Control

Article

Full-text available

Jan 2022

The Institut de Recherche en Sciences de la Santé (IRSS) of Burkina Faso, West Africa, was the first African institution to import transgenic mosquitoes for research purposes. A shift from the culture of mosquito research to regulated biotechnology research and considerable management capacity is needed to set up and run the first insectary for transgenic insects in a country that applied and adapted the existing biosafety framework, first developed for genetically modified (GM) crops, to this new area of research. The additional demands arise from the separate regulatory framework for biotechnology, referencing the Cartagena Protocol on Biosafety, and the novelty of the research strain, making public understanding and acceptance early in the research pathway important. The IRSS team carried out extensive preparations following recommendations for containment of GM arthropods and invested efforts in local community engagement and training with scientific colleagues throughout the region. Record keeping beyond routine practice was established to maintain evidence related to regulatory requirements and risk assumptions. The National Biosafety Agency of Burkina Faso, Agence Nationale de Biosécurité (ANB), granted the permits for import of the self-limiting transgenic mosquito strain, which took place in November 2016, and for conducting studies in the IRSS facility in Bobo-Dioulasso. Compliance with permit terms and conditions of the permits and study protocols continued until the conclusion of studies, when the transgenic colonies were terminated. All this required close coordination between management and the insectary teams, as well as others. This article outlines the experiences of the IRSS to support others undertaking such studies. The IRSS is contributing to the ongoing development of genetic technologies for malaria control, as a partner of Target Malaria (https://targetmalaria.org). The ultimate objective of the innovation is to reduce malaria transmission by using GM mosquitoes of the same species released to reduce the disease-vectoring native populations of Anopheles gambiae s.l.

Advances in Aedes Mosquito Vector Control Strategies Using CRISPR/Cas9

Chapter

Jan 2021

Advancements in genetic engineering have resulted in the development of mosquitoes with impaired vector competence, thereby limiting acquisition and transmission of pathogens. The main dengue (DENV) vector, Aedes aegypti, is an invasive species that have spread unwittingly across the world as a result of human trade and travel. The Ae. aegypti mosquito species has spread across tropical and subtropical regions, with higher presence in urban regions where rapid breeding patterns have shown in artificial containers. Identification of and treating an adequate number of mosquito breeding sites as a control measure have been done for the past couple of years, and yet improvement is far from the expectations, even with well-funded and well-organized initiatives. In order to stop the pathogen transmission, genetically modified mosquitoes (GMM) needs to be created and released. Despite many Aedes-related achievements, GMM creation has been challenging. The spread of particular genetic elements that impair vector competence, trigger deleterious recessive mutations, or skew a population's sex ratio can be used to prevent the spread of vector disease, or eradicate invasive organisms in a species-specific and eco-friendly manner. In recent years, genome editing strategies have evolved to make use of a variety of nucleases, ranging from sequence-specific zinc finger nucleases to modular TALENs (transcription activator-like effector nucleases) and most recently, RNA-guided nucleases adapted from bacterial adaptive immune systems, dubbed CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated systems). By combining these methods, a new era in gene editing had emerged. Generally, both of these gene editing technologies utilize sequence-specific nucleases to generate double-stranded DNA breaks (or nicks) in the target sequence, resulting in desired DNA modifications using endogenous DNA repair mechanisms. Since cells with DNA lesions are unable to divide further, the nuclease-generated strand breaks must be rapidly repaired by the cell to maintain the viability. CRISPR/Cas has been widely accepted for use in a variety of organisms, including insect species, with only minor optimization steps needed thus far. CRISPR/Cas9 technology transformed the process of engineering nucleases capable of cleaving complex genomic sequences. A complementary guide RNA (gRNA) directs the Cas9 endonuclease's operation to the specific DNA target site, enabling the editing of virtually any DNA sequence without complex protein engineering and selection procedures. Apart from genome editing, the specificity and flexibility of the CRISPR/Cas9 method enables unprecedented rapid development of genetically modified organisms with mutation systems for disease vector insect control. The stability and expression of the gene construct generated by CRISPR/Cas9 or any other method must be addressed before GMM are released, in order to make sure that pathogen transmission and formulation are interrupted robustly and completely. Spreading foreign antipathogen genes through gene drive strategies among wild mosquito populations strengthens the case for a more streamlined approach. Major fields that must be adequately assessed include risk evaluation and management, conducting studies to ensure human and environmental protection, developing effective control strategies built on comprehensive gene-driving systems, and adequately addressing the ethical, legal, and social consequences of GMM release. Although GMM is theoretically feasible as a disease control method, field releases should be made only when strong scientific evidence of human and environmental protection and effectiveness are presented, and public acceptance is addressed appropriately. This chapter discusses the diverse technological advances in generating Ae. aegypti mosquitoes which are resistant to dengue virus (DENV) and other diseases, as well as the biosafety and risk assessment of these procedures. Additionally, the chapter outlines a convincing path forward for developing successful genetic-based DENV control strategies based on CRISPR/Cas9, which could be expanded to control other arboviruses while maintaining biosafety.

Infectious Disease Genomics

Chapter

Jan 2024

Yu-Tsueng Liu

Transcriptomics analysis of ethanol treatment of male Aedes aegypti reveals a small set of putative radioprotective genes

Article

Full-text available

Jan 2023

Introduction: Sterile Insect Technique (SIT) is based on releasing sterilized male insects into wild insect populations to compete for mating with wild females. Wild females mated with sterile males will produce inviable eggs, leading to a decline in population of that insect species. Sterilization with ionizing radiation (x-rays) is a commonly used mechanism for sterilization of males. Since irradiation can cause damage to both, somatic and germ cells, and can severely reduce the competitiveness of sterilized males relative to wild males, means to minimize the detrimental effects of radiation are required to produce sterile, competitive males for release. In an earlier study, we identified ethanol as a functional radioprotector in mosquitoes. Methods: Here, we used Illumina RNA-seq to profile changes in gene expression of male Aedes aegypti mosquitoes fed on 5% ethanol for 48 hours prior to receiving a sterilizing x-ray dose, compared to males fed on water prior to sterilization. Results: RNA-seq revealed a robust activation of DNA repair genes in both ethanol-fed and water-fed males after irradiation, but surprisingly few differences in gene expression between ethanol-fed and water-fed males regardless of radiation treatment. Discussion: While differences in gene expression due to ethanol exposure were minimal, we identified a small group of genes that may prime ethanol-fed mosquitoes for improved survivability in response to sterilizing radiation.

Safe Application of Genetically Modified Mosquito (GMM) to Combat Dengue and Chikungunya Depends on Socioeconomic Status and Social Acceptance in the Developing Countries: A Comprehensive Analysis

Chapter

Jan 2021

Mohammad Nazrul Islam

The emerging and re-emerging vector-borne diseases are a serious public health problem throughout the world. It has been observed that more than 100 countries and approximately half of the world’s population are at risk on vector-borne diseases (VBDs). The global burden of the vector-borne diseases is unacceptably high. It alludes toward their functional inappropriateness, untimeliness, and irrelevance in controlling vectors and vector-borne diseases. Modern technologies, coupled with other appropriate ones within the precincts of integrated vector management (IVM), can tide over this situation posed by conventional, mostly insecticide-based, methodologies. A lot of challenges, obstacles, and interruptive factors have warranted urgent deployment of new approaches for the control of VBDs keeping in mind the inbuilt ethical, social, and regulatory issues. Genetically modified mosquito (GMM) technology is a complex and highly sophisticated biotechnological intervention for suppression of vector populations. Wolbachia-associated sterile insect technique (SIT) has been proved highly significant and effective for replacement of mosquito populations. Adopting a highly sophisticated GMM technology to suppress or replace the mosquito populations’ density is a big question in developing countries because their priority is directed to foremost fulfill the basic human rights to sustain. Yet, notwithstanding foreseeable bottlenecks, of paramount importance is the need to deploy GMM technology with due consideration to socioeconomic factors and availability of advanced biotechnological facilities during the application of GMM in the developing countries.

Three Decades of Malaria Vector Control in Sudan: The Plausible Role of Sterile Insect Technique (SIT)

Chapter

Jan 2021

In Northern State, Sudan, a feasibility study for sterile insect technique (SIT) in an area-wide integrated pest management was established for the first time in an African country. The aim of the study was to see whether it is feasible, from a technical, an economical and a biological perspective, to use sterile male mosquitoes to control mosquito populations in designated areas in the African context. The project was focussed on Anopheles arabiensis, one of the major malaria vectors. Meteorological data, larval surveillance and population genetic studies were carried out on the disease vectors. The first phase of the study focussed on the development of an efficient sex-separation system, development of dose-sterility curves for the pupal and adult stages and testing of a range of doses in competition experiments to determine effective sterility dose. This stage was followed by a semi-field phase that monitored their swarming and mating behaviours, effectiveness of irradiated males in competitive experiments with wild males and insemination rates. Information regarding irradiation and transportation of irradiated males were also obtained during the study. Unfortunately, the SIT study was terminated in 2017 before starting field release of irradiated males. In spite of the challenges, such investment need not be totally abandoned as valuable experience has been gained and capacity built, which are of high value to malaria control program in Sudan.

Insect Population Control Using a Dominant, Repressible, Lethal Genetic System

Article

Full-text available

Apr 2000

A major modification to the sterile insect technique is described, in which transgenic insects homozygous for a dominant, repressible, female-specific lethal gene system are used. We demonstrate two methods that give the required genetic characteristics in an otherwise wild-type genetic background. The first system uses a sex-specific promoter or enhancer to drive the expression of a repressible transcription factor, which in turn controls the expression of a toxic gene product. The second system uses non–sex-specific expression of the repressible transcription factor to regulate a selectively lethal gene product. Both methods work efficiently inDrosophila melanogaster, and we expect these principles to be widely applicable to more economically important organisms.

Stable germline transformation of the malaria mosquito Anopheles stephensi

Article

Full-text available

Jul 2000

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element1 and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi , through a transposase-mediated process.

Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti

Article

Full-text available

May 2000

Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.

Mariner transposition and transformation of the yellow fever Mosquito, Aedes aegypti

Article

Mar 1998

The mariner transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito, Aedes aegypti. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified mariner element marked with a wild-type allele of the Drosophila melanogaster cinnabar gene was microinjected into embryos of a kynurenine hydroxylase-deficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a mariner transformation system greatly enhances our ability to study and manipulate this important vector species.

Possible Use of Translocations to fix Desirable Genes in Insect Pest Populations

Article

May 1968

C F Curtis

CHROMOSOME translocation heterozygotes (T/+) are usually semisterile, but translocation homozygotes (T/T) if viable are usually fully fertile. If such a viable translocation were produced in an insect pest, T/T insects could be reared in captivity and released into the wild, where matings with wild types (+/+) would produce T/+ progeny.

Microsatellite Markers and Genotyping Procedures for Anopheles gambiae

Article

Feb 1999
Parasitol Today

The procedures outlined in this article by Rui Wang, Fotis Kafatos and Liangbiao Zheng are well suited to studies of field mosquito populations, and also to the genetic mapping of qualitative and quantitative traits of Anopheles gambiae, a major malaria vector in Africa. An accurate, reproducible and high-throughput microsatellite genotyping procedure has been established.

Distribution of genetic variation among chromosomal forms of Anopheles gambiae s.s.: Introgressive hybridization, adaptive inversions, or recent reproductive isolation?

Article

Mar 2001

A series of four papers in this issue explores the reproductive status of the five chromosomal forms of An. gambiae s.s. using molecular techniques to examine the variation among twelve different genes located throughout the An. gambiae s.s. genome. Results of these and previous studies are consistent with a hypothesis of at least partial barriers to gene flow between some chromosomal forms in the Ivory Coast and other West African countries to the north and west, but introgression between S and M types in Benin and countries to the east. Collectively, these studies indicate the need for a broader geographical sampling of An. gambiae s.s., increased research on mechanisms of prezygotic reproductive isolation and field-based studies of survival and fecundity in hybrids to test for postzygotic reproductive isolation.

Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Article

Mar 2001

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.

Evidence for Recent Population Expansion in the Evolutionary History of the Malaria Vectors Anopheles arabiensis and Anopheles gambiae

Article

Aug 2001

Gene flow in malaria vectors is usually estimated based on differentiation indices (e.g., FST) in order to predict the contemporary spread of genes such as those conferring resistance to insecticides. This approach is reliant on a number of assumptions, the most crucial, and the one most likely to be violated in these species, being mutation-migration-drift equilibrium. Tests of this assumption for the African malaria vectors Anopheles gambiae and Anopheles arabiensis are the focus of this study. We analyzed variation at 18 microsatellite loci and the ND5 region of the mitochondrial genome in two populations of each species. Equilibrium was rejected by six of eight tests for the A. gambiae population from western Kenya and by three tests in eastern Kenya. In western Kenya, all departures from equilibrium were consistent with a recent population expansion, but in eastern Kenya, there were traces of a recent expansion and a bottleneck. Equilibrium was also rejected by two of the eight tests for both A. arabiensis populations; the departure from equilibrium was consistent with an expansion. These multiple-locus tests detected a genomewide effect and therefore a demographic event rather than a locus-specific effect, as would be caused by selection. Disequilibrium due to a recent expansion in these species implies that rates of gene flow, as inferred from differentiation indices, are overestimates as they include a historical component. We argue that the same effect applies to the majority of pest species due to the correlation of their demography with that of humans.

Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element

Article

Jan 2002

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.