Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges
Johns Hopkins University, Bloomberg School of Public Health, Dept of Molecular Microbiology & Immunology, 615 N. Wolfe St, Baltimore, MO 21205-2179, USA
* Author for correspondence (e-mail: mlorena{at}jhsph.edu)
Accepted 17 July 2003
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Summary |
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Key words: Plasmodium, genetic engineering, paratransgenesis, genetic drive mechanisms, genetic sexing, fitness, mosquito
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Introduction |
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Development of the parasite in the mosquito is complex
(Fig. 1; Ghosh et al., 2003) and for
the most part occurs in the midgut (gamete to oocyst stages). Although
thousands of gametocytes are acquired with the blood meal, only a few
successfully mature into oocysts, but each of them produces thousands of
sporozoites (Ghosh et al.,
2001
). Because oocyst formation is a bottleneck in sporogonic
development, targeting pre-sporozoite stages could be a more effective
strategy to block parasite transmission.
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In recent years, methods for the genetic modification of mosquitoes have been developed, and effector genes whose products interfere with Plasmodium development in the mosquito are beginning to be identified. While many of the initial hurdles have been overcome, major questions remain to be answered, foremost among which is how to introduce refractory genes into wild mosquito populations. Here we review strategies to alter mosquito vector competence and consider issues related to translating this knowledge to field applications.
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Strategies to interfere with Plasmodium development in the mosquito |
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Paratransgenesis
Paratransgenesis, the genetic manipulation of commensal or symbiotic
bacteria to alter the host's ability to transmit a pathogen, is an alternative
means of preventing malaria transmission. Bacteria can be engineered to
express and secrete peptides or proteins that block parasite invasion or kill
the parasite in the midgut. This strategy has shown promise in controlling
transmission of Trypanosoma cruzi by Rhodnius prolixus under
laboratory conditions (Beard et al.,
2002). Furthermore, symbiotic bacteria in the tsetse fly have been
isolated, transformed with a reporter gene, and reinserted into the fly
(Beard et al., 1998
). For this
strategy to be used in malaria control, bacteria that can survive in the
mosquito's midgut must be identified. Gram-positive and -negative bacteria,
including Escherichia, Alcaligenes, Pseudomonas, Serratia and
Bacillus, have been identified in the midgut of wild anopheline
adults (Demaio et al., 1996
;
Straif et al., 1998
). These
bacteria are easily cultured in the laboratory and may be suitable targets for
genetic manipulation. Whether these bacteria are stable or transient residents
of the midgut of adult mosquitoes remains to be determined.
To successfully control malaria the refractory proteins or peptides
expressed by the bacteria must act on the midgut stages of the malaria
parasites, maintain their bioactivity in the midgut environment, and be
expressed in sufficient quantities. When An. stephensi mosquitoes
were fed E. coli that express a fusion protein of ricin and a
single-chain antibody against Pbs21 (a P. berghei ookinete surface
protein), oocyst formation was inhibited by up to 95%
(Yoshida et al., 2001). Other
effector molecules, such as SM1 and PLA2, are considered below (see
`Genetically modified mosquitoes'). The use of paratransgenesis for the
control of malaria will require the development of methods to introduce
genetically modified bacteria into field mosquitoes.
Genetically modified mosquitoes
Another promising approach is to genetically modify mosquitoes to express
proteins or peptides that interfere with Plasmodium development.
Methods to produce transgenic culicine
(Jasinskiene et al., 1998) and
anopheline (Catteruccia et al.,
2000
; Grossman et al.,
2001
) mosquitoes are now available
(Fig. 2). Promoters to drive
the transgenes and effector molecules whose products hinder parasite
development are considered below.
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Promoters
An essential step in engineering mosquitoes with reduced vector competence
is the identification of suitable promoters to drive the expression of
anti-parasitic genes. During its development in the mosquito, the parasite
occupies three compartments: midgut lumen, hemocoel and salivary gland lumen.
Thus, promoters that drive synthesis and secretion of proteins into these
compartments need to be identified. In addition to spatial considerations, the
time of protein synthesis relative to arrival of the parasite in each of these
compartments needs to be considered.
As argued above, control of transmission has the best chance of success if
pre-sporozoite stages in the midgut lumen are targeted. Studies in our
laboratory demonstrated that carboxypeptidase, a digestive enzyme, and
AgAper1, a peritrophic matrix protein, are activated in response to a blood
meal and the proteins secreted in the midgut lumen (Edwards et al.,
1997,
2000
;
Moreira et al., 2000
;
Shen and Jacobs-Lorena, 1998
).
Interestingly, transgenes driven by the AgAper1 promoter produce proteins that
are stored in secretory vesicles of midgut epithelial cells prior to a blood
meal and are released immediately after blood ingestion (E. G. Abraham, M.
Donnelly-Doman, H. Fujioka, A. Gosh, L. Moreira and M. Jacobs-Lorena,
unpublished observations). This makes AgAper1 an ideal promoter to target the
earliest stages of parasite development.
Later stages of parasite development can be targeted in the hemocoel and
salivary glands. The vitellogenin promoter and signal sequences were shown to
drive strong gene expression in the fat body and protein secretion into the
hemocoel (Kokoza et al.,
2000). However, this gene has a restricted temporal profile of
expression that peaks around 24 h after a blood meal and returns to basal
level by 48 h. Soon after traversing the midgut epithelium, the ookinete
transforms into an oocyst that is covered by a thick capsule. Sporozoites are
liberated from the oocyst as early as 10 days later. These characteristics of
parasite development limit the choice of effector genes that can be used in
conjunction with the vitellogenin promoter to those encoding proteins with
exceptionally long half-lives. Re-activation of the vitellogenin promoter by
additional blood meal(s) may lessen this shortcoming. The availability of a
strong promoter with peak expression in the hemolymph at about the time of
sporozoite release from oocysts would be ideal. Two salivary gland promoters
have been characterized in transgenic mosquitoes: Maltase-I and Apyrase
(Coates et al., 1999
).
However, expression from both promoters was rather weak, which may limit their
usefulness. Considering that sporozoites can reside in the lumen of the
salivary gland for extended periods, the identification of a strong salivary
gland promoter would be desirable. Finally, mosquito promoters induced by the
presence of the parasite, such as those of immune genes, are of potential
usefulness (Dimopoulos et al.,
2002
; E. G. Abraham, M. Donnelly-Doman, H. Fujioka, A. Gosh, L.
Moreira and M. Jacobs-Lorena, unpublished observations).
Strong and ubiquitous promoters could also be used to drive the expression of effector genes. However, such promoters are not ideal because generalized expression may impose a fitness load on the mosquito (this point is also considered below) and even promoters considered ubiquitous (e.g. actin) are not equally expressed in all cells.
Effector genes
The quest for anti-parasite molecules has been directed towards
identification of gene products that hinder transmission by either killing or
interfering with parasite development. For example, transgenic mosquitoes
expressing defensin, an anti-microbial peptide, effectively killed
gram-negative bacteria, though defensin's action on malaria parasites has not
been documented (Kokoza et al.,
2000). Alternatively, single-chain antibodies that recognize
parasite surface proteins show promise in interfering with parasite
development. As mentioned above, an anti-Pbs21 single chain antibody inhibited
oocyst formation by up to 95% (Yoshida et
al., 2001
). A single-chain antibody against P.
gallinaceum Circunsporozoite protein (CSP), a major surface protein of
sporozoites, expressed from a Sinbdis virus vector, reduced the number of
parasites in the salivary glands by 99%
(de Lara Capurro et al.,
2000
).
Although its mode of action is unknown, phospholipase A2 (PLA2) from a
variety of sources significantly inhibited ookinete invasion of the mosquito
midgut epithelium when mixed with an infected blood meal
(Zieler et al., 2001). Moreira
et al. (2002
) have shown that
transgenic mosquitoes expressing bee venom PLA2 from the carboxypeptidase
promoter reduced P. berghei oocyst formation by 87%. Transgenic
An. stephensi expressing PLA2 from the AgAper1 promoter inhibited
oocyst formation to about the same extent (E. G. Abraham, M. Donnelly-Doman,
H. Fujioka, A. Gosh, L. Moreira and M. Jacobs-Lorena, unpublished
observations).
An alternative strategy is to use synthetic molecules to interfere with
parasite development. Ghosh et al.
(2001) screened a phage
display library for protein domains that bind to the midgut and salivary gland
epithelia and identified a short peptide, named SM1, which inhibited parasite
invasion. Furthermore, An. stephensi engineered with a synthetic gene
expressing a SM1 tetramer under the control of the carboxypeptidase promoter
were impaired in supporting P. berghei development (average 82%
inhibition of oocyst formation). Moreover, in two out of three experiments,
parasite transmission by the transgenic mosquitoes was completely blocked
(Ito et al., 2002
). This
inhibition is thought to occur by peptide binding to epithelial cell surface
proteins (putative receptors) required for parasite invasion. The
effectiveness of SM1 in inhibiting the development of parasites that cause
malaria in humans remains to be demonstrated.
Although major advances have been accomplished in recent years, it is important that the search for new effector molecules and promoters continue for two reasons. First, considering how easily parasites acquire drug resistance, it is likely that parasites will be selected that can overcome the barrier imposed by the effector molecules. Secondly, maximum efficiency of blocking parasite development (ideally 100%) is important for the transgenic mosquito strategy to have a significant impact on disease transmission. Furthermore, while many of the tools for genetic modification of mosquitoes have been developed, an extensive gap exists in our ability to transfer this technology to the field for the control of malaria.
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Challenges facing a successful field release |
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Developing an effective drive mechanism
Two general strategies can be considered for introducing transgenic
mosquitoes in the field: population replacement or a genetic drive mechanism.
Population replacement, or inundatory release, requires a significant
reduction of the resident mosquito population (for instance, with
insecticides), followed by the release of large numbers of refractory
mosquitoes to fill the vacated biological niche. This strategy is promising as
a research tool and as a field test to assess the effectiveness of the
transgenic mosquito approach for interrupting malaria transmission. However,
this strategy cannot be considered for large-scale control purposes, because
it is not possible to produce sufficient numbers of mosquitoes to achieve
population replacement on a country- or continent-wide level.
An efficient genetic drive mechanism is helpful because a manageable number
of genetically modified mosquitoes can replace the wild population, even if
the effector gene(s) imposes some fitness cost. A crucial requirement for this
approach is a tight linkage between the effector gene(s) and the drive
mechanism. Any dissociation between the two would cause the effector gene to
be lost. In contrast to insecticide control, this strategy involves
replacement of one population by another, meaning that in principle, it needs
to be accomplished only once. Transposable elements are an attractive
mechanism to drive genes into populations. Unfortunately, little is known
about their effectiveness. Compelling evidence exists in Drosophila
that transposable elements can efficiently spread through largely distributed
populations. For instance, the P transposable element spread through
the D. melanogaster world population in the short span of about 30
years (Anxolabehere et al.,
1988). Transposable elements such as piggyBac, minos,
mariner and hermes, which have been successfully used to
transform mosquitoes, need to be examined for their usefulness as drive
mechanisms. For example, the piggyBac element is thought to have an
efficient cut-and-paste mechanism and jump nonreplicatively, in a manner
similar to P elements (Lobo et
al., 1999
). When P elements are excised the organism
typically repairs the double stranded break in the chromosome using a
recombinational repair system and uses the uncut chromosome as a template
(Engels, 1997
). In individuals
homozygous for the P element this leads to a duplication of the
element as it transposes to a new location in the chromosome. If a similar
duplication mechanism exists for piggyBac it would act as an
efficient and useful drive mechanism.
Transposable elements may incur a substantial fitness cost. Transposition
causes random integration across the genome, some of which may disrupt genes
and lead to mutations that could be lethal, reduce fecundity or decrease
fitness. Predictive models suggest that transposable elements would be able to
drive refractory genes from a small number of transgenic mosquitoes into the
wild population even if a fitness cost was present
(Ribeiro and Kidwell, 1994;
Boete and Koella, 2003
).
However, there is considerable lack of experimental data to corroborate or
disprove the models. Another consideration is that mobility of the
transposable element may be negatively regulated by a repressor. For instance,
mobility of the P element in D. melanogaster decreases after
several generations because an inhibitor of transposition gradually
accumulates and the fly is said to acquire the P (refractory)
cytotype. This is of practical importance because in such cases the gene(s)
can be driven through a population only once. If the effector gene(s) acquires
mutations or the parasite becomes resistant to the effector gene product
another gene cannot be driven into the same population with the same
transposable element.
A second possible drive mechanism is the use of so-called `selfish genes'.
These genes drive themselves through a population by using the host cell DNA
repair machinery (Burt, 2003).
One such selfish gene, homing endonuclease gene (HEG), encodes an endonuclease
that in heterozygous individuals cleaves the sister chromosome within a 20-30
bp recognition sequence. The chromosome encoding the HEG is not cut because
the selfish gene disrupts the cleavage site. As described for the P
element above, the cell's repair machinery subsequently duplicates the HEG by
using the HEG+ chromosome as a template for recombination. A major
advantage of using HEG as a drive mechanism is that it should allow the
reversal of gene dispersal by introducing an HEG that targets and disrupts the
anti-parasitic gene(s). Furthermore, the possibility of horizontal
transmission is minimized because, unlike transposable elements, the gene is
never excised from the chromosome. However, no precedent exists for the
engineering of homing endonucleases to recognize the desired target sequence.
For a detailed review of selfish genes as drive mechanisms see Burt
(2003
). While on theoretical
grounds the approach is attractive, it is not ready for implementation.
A third potential drive mechanism to introduce an effector gene into a
vector population involves the use of the symbiotic bacterium
Wolbachia, which exerts its drive mechanism primarily through
cytoplasmic incompatibility. When a male infected with Wolbachia
mates with an uninfected female, most of the fertilized eggs perish. However,
when an infected male mates with an infected female, the eggs hatch normally
and the bacteria are transovarially transmitted to the next generation. This
effect can drive Wolbachia through a population very effectively by
giving infected individuals increased reproductive success. For example, a
strain of Wolbachia was discovered in Drosophila simulans
and found to have advanced across the state of California at a rate of 100 km
per year (Turelli and Hoffmann,
1991). Wolbachia may have a wide tissue distribution in
insects, allowing refractory genes to target the various Plasmodium
life stages throughout the mosquito
(Dobson et al., 1999
). Three
methods have been suggested to drive a transgene into a vector population
using Wolbachia: (1) the refractory gene could be introduced directly
into the Wolbachia genome, (2) the genes for cytoplasmic
incompatibility plus the refractory gene could be integrated into the vector's
genome, or (3) the refractory gene could be engineered into a second
maternally transmitted organism that could `hitchhike' with Wolbachia
(Turelli and Hoffmann, 1999
).
Unfortunately, a Wolbachia has yet to be identified in wild
anopheline mosquitoes, although they have been isolated from their culicine
relatives (Ricci et al.,
2002
). If anopheline mosquitoes are physiologically unable to
harbor Wolbachia, identifying the genes conferring cytoplasmic
incompatibility and inserting them into the vector's genome might be feasible.
If however, anophelines tend not to be infected because they have rarely been
exposed to Wolbachia, all three strategies hold potential.
Regardless of the driving mechanism employed, it will be essential that the effector gene (that interferes with Plasmodium development) be tightly linked to the driving element. A dissociation of the two (for instance, by recombination) will cause the driving gene to continue to spread through the population alone while the effector gene is lost.
Mass production of transgenic insects and genetic sexing
mechanisms
Transgenic-based methods to reduce or eradicate vector populations, such as
the release of insects carrying a dominant lethal (RIDL;
Thomas et al., 2000), show
promise for some species. However, their use as a malaria control program in
Africa would be difficult to implement due to reproductively incompatible
subspecies and migration of mosquitoes among villages. Even if successful,
this approach would leave an ecological vacuum that another malaria vector
could quickly fill. Therefore, replacement of wild populations with transgenic
mosquitoes carrying refractory genes instead of population suppression or
eradication methods would be more appropriate.
Unfortunately, this approach still requires the release of vast numbers of biting insects, which is ethically questionable due to their nuisance factor and potential role as vectors for secondary diseases. Thus, widespread release of genetically modified mosquitoes is best done using only non-biting males, necessitating an efficient system for male selection. Moreover, the ability to release only males would provide a more realistic prospect of making the use of transgenic mosquitoes acceptable to the local communities and to the public in general.
Males of a few insect species can be separated by physical methods, based
on sexual dimorphism (Sharma et al.,
1976; Alphey and Andreasen,
2002
). However, these techniques do not easily translate to most
vector species, including anopheline mosquitoes, and rarely give 100% sex
separation. An alternative approach is to use genetic techniques for
sex-sorting, known generically as genetic sexing mechanisms (GSMs). It allows
males and females to be produced under one set of conditions to allow
propagation and males only under selective conditions. GSMs based on the
radiation-induced translocation of semi-dominant genes for insecticide
resistance onto the Y chromosome were developed in An. gambiae and
Anopheles albimanus (Curtis et
al., 1976
; Seawright et al.,
1978
). This system was used for the production of a million
An. albimanus sterile males per day
(Dame et al., 1981
), but
introducing insecticide resistance into the wild populations is too risky for
field applications. Moreover, genetic crossing over between the relevant genes
may occur, disrupting the production of a single sex population
(Curtis, 2002
).
Transgene-based GSMs using conditional female-specific lethality systems,
based on the tetracycline-repressible expression system of Gossen and Bujard
(1992), show great promise and
were successfully tested in Drosophila
(Thomas et al., 2000
;
Heinrich and Scott, 2000
). In
this system, two genes are engineered into the insect: one that expresses a
tetracycline-repressible transcriptional activator protein (tTA), and a tetO
promoter element linked to a lethal gene. In the absence of tetracycline, tTA
binds to tetO leading to expression of the lethal gene and death of the
insect. When tetracycline is added to the diet tTA fails to activate the tetO
promoter and allows the insects to survive and propagate. Female-specific
lethality can be achieved in two ways: the lethal gene itself can be
female-specific, or a female-specific promoter can be used to drive tTA
expression, in turn inducing expression of the toxic gene. Unfortunately,
using a repressible system to generate males for a large-scale field release
would incur a massive fitness cost, since refractory females possessing the
female-specific lethality system would be immediately killed in the wild and
thus would not contribute to the gene pool. For this reason, a better approach
would be to use an inducible system rather than a repressible one.
An inducible female-specific lethality system prevents expression of the
lethal gene in the absence of induction, allowing male selection prior to
release, but permitting subsequent generations of females to survive in the
wild. A tetracycline-inducible expression system developed by Baron and Bujard
(2000) has the same components
as the tetracycline-repressible expression system, except a mutated version of
tTA that binds to tetO only induces the expression of the lethal effector gene
in the presence of tetracycline.
Avoiding resistance to the refractory genes
Parasites facing a refractory mosquito population would be under strong
selective pressure, similar to the one posed by anti-malarials, and thus
resistance may develop. Engineering a mosquito with multiple refractory genes
that target different aspects of parasite development could minimize
resistance to the refractory genes. For example, a transgenic mosquito might
be engineered to express a peptide to disrupt midgut and salivary gland
invasion, have an enhanced encapsulation response to target the oocyst, and
express defense peptides to target the sporozoites. Furthermore, chances of
success will be greatly increased if each refractory element is as close to
100% effective as possible and if introduction of the refractory genes is
coupled with traditional control methods, such as reduction of wild
populations with insecticides prior to a transgenic release, drug treatment of
infected individuals, and use of bed nets.
The effectiveness of transposable elements may decrease with time after
field release. Immediately after the introduction of a novel transposable
element into a population the element enjoys a period of unrestrained activity
and spreading. Eventually, individuals with mutations in the transposase or
those that have enacted regulatory inactivation of the element will be
selected. Transposase silencing has been well studied in the mariner family
and has been hypothesized to occur by several mechanisms, including
overproduction inhibition whereby an increase in transposase activity
correlates with decreased transposition or random transposase mutations.
Random transposase mutations may lead to open reading frame disruptions and
inactive transposases that compete with active transposase for substrate
(competitive inhibition) or reduce the activity of wild-type transposase
(dominant negative complementation; Hartl et al.,
1997a,b
;
Tosi and Beverley, 2000
). The
mechanism of transposable element silencing will need to be well understood
before transposable elements are used in the field.
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Conclusions |
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