Opinion TRENDS in Parasitology
5 May 2006
Mosquito transgenesis: what is the
fitness cost?
Mauro T. Marrelli1*, Cristina K. Moreira1*, David Kelly2,3*, Luke Alphey2,3 and
Marcelo Jacobs-Lorena1
1
Johns Hopkins University, Bloomberg School of Public Health Department of Molecular Microbiology and Immunology and
Malaria Research Institute, 615 North Wolfe St, Baltimore, MD 21205, USA
2
Department of Zoology, University of Oxford, South Parks Road, Oxford, UK, OX1 3PS
3
Oxitec Ltd, 71 Milton Park, Oxford, UK, OX14 4RX
The generation of transgenic mosquitoes with a mini-
mal fitness load is a prerequisite for the success of
strategies for controlling mosquito-borne diseases
using transgenic insects. It is important to assemble as
much information as possible on this subject because
realistic estimates of transgene fitness costs are
essential for modeling and planning release strategies.
Transgenic mosquitoes must have minimal fitness
costs, because such costs would reduce the effective-
ness of the genetic drive mechanisms that are used to
introduce the transgenes into field mosquito popu-
lations. Several factors affect fitness of transgenic
mosquitoes, including the potential negative effect of
transgene products and insertional mutagenesis.
Studies to assess fitness of transgenic mosquitoes in
the field (as opposed to the laboratory) are still needed.
Genetic manipulation of mosquitoes for disease control
Mosquitoes are vectors of serious human infectious
diseases, such as malaria, dengue and yellow fever.
From the 1950s to the 1970s there was considerable
optimism that such diseases could be controlled or even
eradicated by the use of insecticides and drugs. However,
these expectations were not realized, in part because of
increasing mosquito resistance to insecticides, parasite
resistance to drugs and slow progress in vaccine develop-
ment. Genetic modification of mosquitoes has been
proposed as an alternative strategy for disease control
[1–3].
Control at the level of the insect vector can be divided into
two broad categories. The first is the genetic modification of
mosquito populations, which could be achieved by the
release of transgenic mosquitoes carrying genes whose
products impair pathogen development. This approach
requires the use of a special genetic system capable of
spreading the anti-pathogen gene of interest through a
target vector population. Such genetic drive mechanisms
have not yet been realized; various molecular systems, for
example, transposable elements, have been proposed as the
basis for such a system [4] The second is population
suppression, which could be achieved by use of the sterile
Corresponding author: Jacobs-Lorena, M. (mlorena@jhsph.edu).
* These authors contributed equally to this publication.
Available online 24 March 2006
www.sciencedirect.com 1471-4922/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2006.03.004
Vol.22 No.
insect technique (SIT) [5] in conjunction with a technology
known as ‘release of insects carrying a dominant lethal’
(RIDL) [6]. In RIDL, insects are transformed with a
transgene whose product suppresses offspring production,
leading to a decrease of the vector population.
The prospect of using transgenic mosquitoes is rapidly
gaining strength, owing to the identification of transposable
elements for mosquito germ line transformation, the finding
of suitable transformation markers such as fluorescent
proteins [7], the standardization of microinjection tech-
niques [8–10], the characterization of promoters that can
drive the expression of foreign genes in a tissue- and stage-
specific manner [11–15] and the identification and charac-
terization of effector molecules that can interfere with the
development of parasites in the invertebrate host. Effector
molecules include naturally occurring or synthetic anti-
microbial peptides, antibodies against parasite or mosquito
midgut proteins and proteins with toxic or inhibitory effects
[16–27], or molecules that can interfere with the develop-
ment of the invertebrate host itself [28].
One crucial issue that needs to be considered before the
release of genetically manipulated organisms is the fitness
of the mosquitoes carrying the transgene. This is because
the transgenic insects must compete effectively with the
local populations to efficiently introgress the effector genes
into the wild gene pool. Fitness can be defined as the
relative success with which a genotype transmits its genes
to the next generation. It has two major components,
survival and reproduction, and can be evaluated by
analyzing several parameters, such as fecundity, fertility,
larval biomass productivity, developmental rate, adult
emergence, male ratio, and mating competitiveness.
Additional factors that can reduce fitness are inadequate
mass rearing conditions, inbreeding and hybridization
with mosquitoes of different genetic backgrounds. Other
issues, such as drive mechanisms, have been reviewed
elsewhere (e.g. Refs [3,4]). Here, we focus on the possible
fitness costs of transgenesis and draw some lessons for
future studies of fitness of genetically
manipulated mosquitoes.
Potential fitness costs of transgenesis
The potential fitness impact of transposon-mediated
transgenesis can be broadly divided into the negative
198 Opinion TRENDS in Parasitology
effect of transgene products and insertional mutagenesis
after a transposition event.
Burden from the transgene product
Transgenic insects typically express multiple genes; for
example, a fluorescent marker and an anti-pathogen
effector protein [17,29,30]. Constructs for RIDL encode,
in addition to the marker and effector, a repressible
transactivator protein for tight control of the system
(reviewed in Ref. [31]). The accumulation of foreign
proteins might be toxic to the cells in which they are
expressed. Proteins expressed in a restricted cell type are
less likely to have an impact on fitness than ubiquitously
expressed proteins. For instance, fluorescent protein
expression in the eye does not appear to affect fitness, at
least not in the laboratory [32]. Whether or not fluorescent
protein expression affects vision and fitness in the field
remains to be determined. Proteins expressed from strong
and ubiquitous promoters (e.g. the actin promoter) might
cause a fitness load as a result of accumulation of large
amounts of foreign proteins in a wide variety of cell types
[33]. In some cases, proteins that accumulate in the cell
cytoplasm can have a greater impact on fitness than
secreted proteins, because the concentrations reached in
the former case are likely to be much higher.
Of course, the nature of the protein itself is a crucial
factor for fitness. For instance, although no effect on
fitness was observed for mosquitoes expressing the 12
amino acid peptide SM1, mosquitoes expressing phospho-
lipase A2 (PLA2) were clearly less fit and less fertile than
0.40
0.35
0.30
0.25
Frequency
0.20
0.15
0.10
0.05
0
0.78 1.56 3.13 6.25
Homozygous viability as a percentage of heterozygous viability (log scale)
Figure 1. Frequency distribution of the viability of 706 homozygous single P element insertion lines of Drosophila melanogaster, as a percentage of heterozygote viability.
Adapted from data in Figure 4A of Ref. [38].
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Vol.22 No.5 May 2006
wild type [32]. Decreased fitness was probably caused by
damage to midgut epithelial cells [14], as no adverse effect
was observed when PLA2 was administered per os [25].
Toxicological effects are more of a concern with the RIDL
system, which, after all, is designed to selectively kill a
targeted subpopulation of individuals (e.g. females only).
Thus, constructs should be engineered to reduce possible
harm to the non-targeted subpopulation (e.g. males) by
choosing promoters with low leaky basal expression, and
effector proteins (the ones that do the killing) that act in a
measured, stoichiometric manner, rather than in a run-
away, catalytic manner in which even small quantities of
effector protein might be toxic. Furthermore, the effector
gene can be engineered to be expressed only in relevant
tissues and at specific life stages. This can be achieved by
choosing tissue- and stage-specific promoters to drive the
expression of the effector genes. For example, a RIDL
effector gene might be driven by an embryo-specific
promoter (successfully tested in Drosophila melanogaster
[34]), whereas an anti-malaria effector might be driven by
either the midgut-specific and blood-meal-inducible
carboxypeptidase promoter [17,29,30] or the female gut-
specific peritrophic matrix protein 1 (AgAper1) promoter
and its associated regulatory sequences; these sequences
promote storage of the protein (in addition to mRNA) in the
midgut epithelium before blood ingestion [14]
Insertional mutagenesis
The second way in which transgenesis might reduce
fitness is through disruption of native gene function.
12.5 25 50 100 200
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 Opinion TRENDS in Parasitology Vol.22 No.5 May 2006 199

Insertion of transgenes might occur in transcriptionally
active areas of the genome [35–37]. In an extreme
situation, insertion of a transposon into an essential
gene would prevent expression of the functional gene
product. However, for most genes, disruption is likely to be
recessive (no phenotype detected in heterozygotes), and
disruption of an essential gene would be lethal only in
homozygotes. Most insertions seem to have little or no
effect on fitness, presumably because they either integrate
into regions of the genome that do not encode genes or do
not significantly disrupt native gene function [38].
Limited experience with mosquitoes and other insects
in our laboratories suggests that fitness reduction through
insertional mutagenesis is not frequent. More extensive
studies of the effect of insertional mutagenesis in
Drosophila provide us with some insights into the issue.
In one of the best studies of its kind, Lyman et al. [38]
measured the fitness costs of 706 independent
D. melanogaster lines carrying single P-element inser-
tions. The host strain for the mutagenesis was an inbred
Samarkand (Sam) strain, which had been maintained for
over 100 generations of continuous full-sib inbreeding and
was therefore essentially isogenic. The fitness conse-
quences of insertional mutagenic effects could thus be
observed in the absence of any ‘hitchhiking’ effects
(fixation of recessive deleterious genes near the point of
transgene insertion). Each transgenic line was generated
by insertion of a P element marked with an eye color gene
(rosy, ryC). To assess fitness, each independent trans-
formed line was grown up from embryos as a heterozygote
or a homozygote in the presence of (i.e. in competition
with) the wild type, and the ratio of wild type to
transformed adults that emerged was scored. These
measurements are particularly relevant to questions of
productivity of mass-reared transgenic insects and might
also reflect several other fitness parameters, such as adult
male mating success.
The ryC marker appears to improve the viability of
Drosophila: on average, the heterozygous lines were fitter
than the wild type. This means that the absolute effect of
the insertional mutagenesis of the transposon on viability
cannot be measured relative to the wild-type control.
Lyman et al. [38] demonstrate that the insertional
mutagenic effects on viability are recessive. We can
therefore examine the viability consequences of P-element
insertions in homozygotes, relative to heterozygotes from
the same line (Figure 1).
Two conclusions can be drawn from these data. First,
that the change in viability is almost always zero or
negative: this makes biological sense, because any random
insertion (or other mutation) is likely to be deleterious.
Instances of increased fitness in the data of Lyman et al.
[38] could be attributed to the relatively low resolution of
the assay and to the potential for insects homozygous for
ryC to have an advantage over those heterozygous for ry.
The lack of dramatic increase in fitness should reassure
regulators that transgenic insects are unlikely to inad-
vertently acquire a selective advantage through inser-
tional mutagenesis. However, the potential loss of fitness
caused by insertional mutagenesis reinforces the need for
mechanisms to drive genes whose products impair
pathogen development through wild insect populations.
The second conclusion is that the distribution suggests
that the fitness of transgenic lines is a probabilistic event,
and there is a reasonable chance of producing one with
only a modest penalty by producing multiple lines and
selecting the fittest. A ‘modest’ penalty would have
different implications for different strategies. For SIT,
reduction in fitness of the released insect is compensated
for by release of larger numbers. This is already the case
for existing SIT programs, in which the number of
transgenic insects released must be up to 100 times the
number of wild insects in the area treated, to compensate
for fitness reduction as a result of mass-rearing conditions,

Table 1. Transgenic mosquitoes lines used for fitness studies

Line Species Transpo- Promoter Tissue Activation Gene Kept as Fitness Refs
son products load
IV An. stephensi Minos Actin5c Ubiquitous Constitutive EGFP Homozygous Yes [40]
hsp70 Ubiquitous Leaky Hygromicin
resistance
VD12 An. stephensi Minos Actin5c Ubiquitous Constitutive EGFP Homozygous Yes [40]
hsp70 Ubiquitous Leaky Hygromicin
resistance
AsML12 An. stephensi Minos Actin5c Ubiquitous Constitutive EGFP Homozygous Yes [40]
Antryp 1P Midgut ? Luciferase
MinRED1 An. stephensi Minos Actin5c Ubiquitous Constitutive DsRed Homozygous Yes [40]
EGFP Ae. aegypti Hermes Actin5c Ubiquitous Constitutive EGFP Homozygous Yes [41]
autoHermes Ae. aegypti Hermes Actin5c Ubiquitous Constitutive EGFP Homozygous Yes [41]
hsp70 Ubiquitous Leaky Transposase
pBacMOS Ae. aegypti piggyBac 3xP3 Eyes Constitutive EGFP Homozygous Yes [41]
hsp70 Ubiquitous Leaky MOS1
transposase
CCBF6 An. stephensi piggyBac 3xP3 Eyes Constitutive EGFP Heterozygous No [29,30,32]
Carboxy- Midgut Blood- SM1
peptidase Epithelium inducible
CCBF3 An. stephensi piggyBac 3xP3 Eyes Constitutive EGFP Heterozygous No [29,30,32]
Carboxy- Midgut Blood- SM1
peptidase Epithelium inducible
PLA2 An. stephensi piggyBac 3xP3 Eyes Constitutive EGFP Heterozygous Yes [29,30,32]
Carboxy- Midgut Blood- PLA2
peptidase Epithelium inducible

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200 Opinion TRENDS in Parasitology
radiation-sterilization and the rigors and inefficiencies of
the release protocols. For driving genes into wild
populations, fitness reductions must be compensated for
by the development of a gene driver with sufficient force to
overcome the fitness deficit [4,39].
Assessing the fitness of transgenic mosquitoes
Three studies have recently examined the impact of
transgenes on the fitness of genetically modified mosqui-
toes [32,40,41] (Table 1). In the first, Catteruccia et al. [40]
evaluated the ability of transgenic Anopheles stephensi to
compete with wild-type mosquitoes of the same species.
Equal numbers of transgenic and non-transgenic mosqui-
toes were mixed in cages and the frequency of the
transgene was followed for several generations. In all
four lines examined, the transgenic allele frequency
decreased sharply until extinction. Analysis of the
integration site of the transgene in two lines (which
carried the same construct) showed that the transposon
had disrupted the coding sequence of a non-essential gene
in one of the lines and that this line had a significantly
lower fitness than the other, in which the transgene did
not disrupt a gene.
In the second study, Irvin et al. [41] examined the
reproductive and developmental fitness of three trans-
genic lines of Aedes aegypti relative to non-transgenic
mosquitoes. Their results showed that all lines analyzed
had high fitness costs. Survivorship was significantly
reduced for all life stages and a higher mortality rate for
the transition from egg to larva was observed. Moreover,
fecundity was considerably decreased and adult longevity
was lower in two lines. In one of the lines an active Hermes
transposase catalyzed somatic transposition of a Hermes
element, and this could have had deleterious effects.
In the third study, the fitness of mosquitoes carrying
two different transgenes that render them unable to
(a)
(b)
TRENDS in Parasitology
Figure 2. The hitchhiking effect. (a) A chromosomal region hemizygous for a
transgene insert (green triangle); (b) a region homozygous for the transgene insert.
Most organisms carry numerous recessive mutations that affect fitness and are
found throughout the genome (red boxes). As the integration of transgenes is
random, they will insert with a certain probability in the vicinity of such recessive
mutations. In such cases, when the transgene is made homozygous, any nearby
recessive gene will also become homozygous (the hitchhiking effect) and fixed,
exerting its effect on fitness. For this reason, it is best to use heterozygous
mosquitoes to assay fitness load that is due to the transgene. Black boxes represent
genes with no fitness load.
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Vol.22 No.5 May 2006
transmit Plasmodium berghei was evaluated [32]. All
lines had the same marker and the same promoter driving
the expression of two different anti-parasitic effector
proteins. One effector was a tetramer of the dodecapeptide
SM1 [24,29] and the other was PLA2 [25,30]. SM1 is
believed to compete with the parasite for binding to a
midgut receptor, whereas the mechanism of PLA2
inhibition is unknown. Notably, the SM1 lines had no
detectable fitness load relative to the non-transgenic
mosquito controls. Conversely, the PLA2 lines competed
poorly with non-transgenics in cage experiments, and the
transgenic allele almost disappeared by the fifth gener-
ation owing to decreased fecundity.
An important difference between the first two of these
studies and the third is that Catteruccia et al. [40] and
Irvin et al. [41] maintained the transgenic lines as
homozygotes, whereas Moreira et al. [32] maintained
their lines as heterozygotes, by crossing at each gener-
ation transgenic mosquitoes to wild-type mosquitoes from
laboratory population cages. The latter strategy ensured
that the genetic background of the transgenic lines was
the same as that of the control mosquitoes, allowing a
more direct correlation between fitness load and the
presence of the transgene. The decreased fitness observed
for the homozygous transgenic mosquitoes can be inter-
preted in two ways: (i) decreased fitness is a consequence
of either negative effects of the transgene product or
‘insertional mutagenesis’ during transgene transposition;
or (ii) decreased fitness is a consequence of the hitchhiking
effect (Figure 2). The two possibilities cannot be
distinguished from the experiments conducted in the
first two studies [40,41]. The fact that no fitness cost
was observed in the two independent SM1 lines in the
third study [32] suggests that transgenesis per se is not
always deleterious.
Several lessons can be derived from the published
studies [32,40,41]. An important one is that inbreeding
can have a strong impact on fitness. Most organisms carry
numerous recessive mutations that reduce fitness [42,43],
and fixation of such alleles will cause inbreeding
depression (Figure 2). The use of heterozygotes to assess
the impact of the transgene itself (as opposed to other
factors) on fitness should be considered for two reasons.
First, heterozygotes are not subject to the inbreeding
effect. Second, the Drosophila literature suggests that
mutations caused by insertional mutagenesis are usually
recessive [38] and would not be detected in heterozygotes.
However, for practical applications such as mass rearing
for field release, homozygous lines are required; it is thus
desirable to choose fittest homozygous lines. As inser-
tional mutagenesis is a probabilistic event, the fitness of
multiple transgenic lines of the same construct should be
compared (as in Figure 1) and the fittest one chosen. When
a hitchhiking effect is suspected, outbreeding the trans-
genic line for multiple generations might improve fitness,
because nearby deleterious allele(s) could be genetically
removed as outcrossing progresses.
When constructing transgenic lines, tissue-specific
promoters (e.g. an eye-specific promoter for marker
genes or a gut-specific or fat body-specific promoter for
effector genes) should be favored over strong and
 Opinion TRENDS in Parasitology
ubiquitously expressed promoters. Strong and ubiquitous
promoters lead to the accumulation of abundant foreign
protein in many cell types, and this could have deleterious
effects [33]. Finally, strength of transgene expression can
be influenced by chromatin surrounding the site of
insertion (position effects). Thus, when strength of
expression is important, several independently obtained
lines for the same construct should be compared.
Alternatively, the use of insulator elements could
be considered.
Plasmodium infection and mosquito reproductive
fitness
Malaria parasites have been reported to cause significant
penalties on the reproductive fitness of mosquitoes
(reviewed in Ref. [44]). In infected mosquitoes, a
proportion of developing oocytes are resorbed following
follicle cell apoptosis, and the total yolk content in the
ovaries is reduced compared with the wild type, leading to
a significant decrease in the number of eggs laid [45,46].
Interestingly, this phenomenon seems to be independent
of parasite density, as it is also observed in the field, where
infections are very low (less than five oocysts per
midgut) [47].
It has been hypothesized that, in the field, genes
associated with natural mosquito resistance to the
malaria parasite might confer a fitness advantage [44].
Therefore, it is expected that mosquitoes carrying a
transgene whose product interferes with Plasmodium
development would have a competitive advantage over
their non-transgenic counterparts when fed on Plasmo-
dium-infected blood. To address this hypothesis, we have
conducted cage experiments similar to those by Moreira
et al. [32], in which a starting population of equal numbers
of wild type and SM1 transgenic mosquitoes was
maintained by feeding at every generation with
P. berghei-infected blood (M.T.M. and M.J-L., unpub-
lished). We observed that under these conditions, the
transgenic mosquitoes had a clear advantage over non-
transgenics with the same genetic background, reaching a
prevalence of w70%. These results suggest that
expression of a transgene that interferes with Plasmo-
dium development can increase the fitness of mosquitoes
that ingest an infected blood meal. While such a fitness
advantage is beneficial to the mosquito, in the field the
impact of the advantage is likely to be very small and not
sufficient on its own to promote introgression of an effector
gene. This is because the rate of mosquito infections, even
in highly endemic areas, is very low [48,49].
Perspectives
The genetic manipulation of mosquitoes for the control of
vector-borne diseases has reached a stage at which
implementation of transgenic insect strategies can now
be considered a realistic prospect. Ideally, one would like
to generate transgenic mosquitoes that are equally (or
better) capable of surviving and mating with their wild-
type counterparts than are those wild-type insects. It is
important to assemble as much information as possible on
this subject because realistic estimates of transgene
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Vol.22 No.5 May 2006 201
fitness costs are essential for modeling and planning
release strategies.
Studies of other transgenic organisms give us some
general hints about the impact of transgenes on fitness,
but, they do not measure certain traits that are specific for
survival in the field. Drosophila studies, as well as the
mosquito cage experiments reported so far, provide good
initial information, but there is need to develop new
assays for dispersal, courtship, mating, ejaculate quality,
sperm competition and longevity of transgenic mosquitoes
in the field. Fitness of transgenic mosquitoes must be
compared not only with non-transgenic mosquitoes from
laboratory colonies but also with outbred mosquitoes that
resemble wild mosquito populations as closely as possible.
Moreover, finding good indicators for fitness (e.g. body
size, wing length or longevity) would be very helpful for
choosing preferred lines from multiple transgenic ones
before progressing to field trials.
In the field, natural selection will act on transgenic
organisms as it does on all others [50]. Therefore, for a
transgene to persist and spread in the wild, transgenic
mosquitoes must have fitness advantages to compensate
for any costs that might be associated with the transgene.
Transgenic organisms with increased fitness would be
selected positively and the transgenic allele would be
spread and fixed in the wild population. Alternatively, the
transgenes must be tightly linked to a genetic drive
mechanism sufficiently strong to overcome the fitness
load. Regardless of the strategy employed, the chances of
success and efficiency of the control measures will increase
as the fitness of the transgenic mosquito increases.
Acknowledgements
Work in the authors’ laboratory was supported by grants from the
National Institutes of Health.
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