Mosquitos Transgenicos

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Vol.22 No.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 minimal 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 effectiveness of the genetic drive mechanisms that are used to introduce the transgenes into field mosquito populations. 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 development. 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. ([email protected]). * These authors contributed equally to this publication. Available online 24 March 2006

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 techniques [8–10], the characterization of promoters that can drive the expression of foreign genes in a tissue- and stagespecific manner [11–15] and the identification and characterization of effector molecules that can interfere with the development of parasites in the invertebrate host. Effector molecules include naturally occurring or synthetic antimicrobial peptides, antibodies against parasite or mosquito midgut proteins and proteins with toxic or inhibitory effects [16–27], or molecules that can interfere with the development 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

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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 phospholipase A2 (PLA2) were clearly less fit and less fertile than

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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 runaway, 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 gutspecific 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.

0.40

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0 0.78

1.56

3.13

6.25

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50

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Homozygous viability as a percentage of heterozygous viability (log scale) TRENDS in Parasitology

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]. www.sciencedirect.com

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Vol.22 No.5 May 2006

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 insertions. 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 consequences 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 transformed 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.

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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 inadvertently acquire a selective advantage through insertional 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

Promoter

Tissue

Activation

An. stephensi

Transposon Minos

IV

Actin5c hsp70

Ubiquitous Ubiquitous

Constitutive Leaky

VD12

An. stephensi

Minos

Actin5c hsp70

Ubiquitous Ubiquitous

Constitutive Leaky

AsML12

An. stephensi

Minos

MinRED1 EGFP autoHermes

An. stephensi Ae. aegypti Ae. aegypti

Minos Hermes Hermes

pBacMOS

Ae. aegypti

piggyBac

Actin5c Antryp 1P Actin5c Actin5c Actin5c hsp70 3xP3 hsp70

Ubiquitous Midgut Ubiquitous Ubiquitous Ubiquitous Ubiquitous Eyes Ubiquitous

Constitutive ? Constitutive Constitutive Constitutive Leaky Constitutive Leaky

CCBF6

An. stephensi

piggyBac

CCBF3

An. stephensi

piggyBac

PLA2

An. stephensi

piggyBac

3xP3 Carboxypeptidase 3xP3 Carboxypeptidase 3xP3 Carboxypeptidase

Eyes Midgut Epithelium Eyes Midgut Epithelium Eyes Midgut Epithelium

Constitutive Bloodinducible Constitutive Bloodinducible Constitutive Bloodinducible

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Gene products EGFP Hygromicin resistance EGFP Hygromicin resistance EGFP Luciferase DsRed EGFP EGFP Transposase EGFP MOS1 transposase EGFP SM1

Kept as

Refs

Homozygous

Fitness load Yes

Homozygous

Yes

[40]

Homozygous

Yes

[40]

Homozygous Homozygous Homozygous

Yes Yes Yes

[40] [41] [41]

Homozygous

Yes

[41]

Heterozygous

No

[29,30,32]

EGFP SM1

Heterozygous

No

[29,30,32]

EGFP PLA2

Heterozygous

Yes

[29,30,32]

[40]

200

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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 mosquitoes [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 mosquitoes 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 transgenic 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)

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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. www.sciencedirect.com

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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 generation 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 generation 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 interpreted 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 insertional 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 transgenic 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

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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 Plasmodium-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., unpublished). We observed that under these conditions, the transgenic mosquitoes had a clear advantage over nontransgenics with the same genetic background, reaching a prevalence of w70%. These results suggest that expression of a transgene that interferes with Plasmodium 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 wildtype 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 www.sciencedirect.com

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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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