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Am. J. Trop. Med. Hyg., 76(6), 2007, pp. 1072-1078
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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GENETIC CONTROL OF MALARIA PARASITE TRANSMISSION: THRESHOLD LEVELS FOR INFECTION IN AN AVIAN MODEL SYSTEM

NIJOLE JASINSKIENE, JUDY COLEMAN, AURORA ASHIKYAN, MICHAEL SALAMPESSY, OSVALDO MARINOTTI, AND ANTHONY A. JAMES*
Department of Molecular Biology and Biochemistry, University of California, Irvine, California; Department of Microbiology and Molecular Genetics, University of California, California


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic strategies for controlling malaria transmission based on engineering pathogen resistance in Anopheles mosquitoes are being tested in a number of animal models. A key component is the effector molecule and the efficiency with which it reduces parasite transmission. Single-chain antibodies (scFvs) that bind the circumsporozoite protein of the avian parasite, Plasmodium gallinaceum, can reduce mean intensities of sporozoite infection of salivary glands by two to four orders of magnitude in transgenic Aedes aegypti. Significantly, mosquitoes with as few as 20 sporozoites in their salivary glands are infectious for a vertebrate host, Gallus gallus. Although scFvs hold promise as effector molecules, they will have to reduce mean intensities of infection to zero to prevent parasite transmission and disease. We conclude that similar endpoints must be reached with human pathogens if we are to expect an effect on disease transmission.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The feasibility of using genetic-based control technologies to limit the size of vector populations (population reduction) or to alter the populations so that they are resistant to pathogens (population replacement) has been researched intensely during the last years.1,2 Success of the population replacement strategy is contingent in part on engineering effector genes that impair replication or development of the pathogens within the vectors.3,4 Among the classes of effector mechanisms being tested are those that interact with putative pathogen ligands.3 Toward this end, we developed single-chain antibodies (scFv) that bind malaria parasites.5 The monoclonal antibody (MAb), N2H6D5, from a mouse hybridoma line, recognizes the circumsporozoite protein (CSP) of the avian malaria parasite, P. gallinaceum, and blocks sporozoite invasion of Ae. aegypti salivary glands when injected into infected mosquitoes.6 A marked reduction of mean intensities of infection of sporozoites in the salivary glands also was observed in mosquitoes expressing transiently an scFv derived from N2H6D5 (N2scFv).5 Here we describe efforts to block malaria parasite development in stable transgenic mosquitoes expressing N2scFv. These analyses confirm that this molecule is effective in inhibiting sporozoite penetration of the salivary glands. Furthermore, our important finding is that the probing and feeding of mosquitoes harboring small numbers of salivary gland sporozoites (20–50) infects vertebrate hosts. The epidemiologic implications of this finding are significant for the proposed transgenic mosquito-based population replacement method for controlling malaria transmission.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mosquito rearing and maintenance. The Higgs white-eye and RED strains of Ae. aegypti,7 both susceptible to infection with P. gallinaceum, were used in the experiments. Mosquitoes were reared using standard laboratory procedures.8 Briefly, larvae were fed on finely ground fish food (Tetramin) (Doctors, Foster and Smith, Rhinelander, WI) and adults fed on 10% sucrose and water ad libitum. Female mosquitoes were fed on anesthetized chickens (Gallus gallus) when necessary. All stages were reared at 27°C, 80% humidity, and a 16-hour/8-hour light/dark photoperiod.

Transformation vectors. Three distinct plasmids containing the N2scFv open reading frame (ORF) were constructed (Figure 1Go). The transgenes were designed to test the accumulation and efficacy of N2scFV after expression directed by the promoters derived from two genes, Ae. aegypti vitellogenin-1 (Vg1)9 or Drosophila melanogaster polyubiquitin (Ub).10,11 Furthermore, the effects on end-product abundance were evaluated by the use of two distinct 3'-end untranslated regions (3'UTRs) derived from the Ae. aegypti defensin gene DefA12 or the SV40 polyadenylation signal. All constructs had a DNA sequence encoding the Maltase-like (Mal I)13 signal peptide included immediately adjacent to the 5'-end of the N2sFV ORF to direct secretion of the effector molecule into the mosquito hemolymph.


Figure 1
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  		FIGURE 1. Schematic representations of donor constructs used to genetically transform Ae. aegypti. The directions of transgene transcription are shown by the small arrows. A, VgN2SV. B, VgN2DF. C, UbN2SV. Large block arrows represent the right (MR, HR) and left (ML, HL) terminal inverted repeats of the Mos1 and Hermes transposable elements. All constructs carry the coding sequences of the EGFP gene to provide a visible transformation marker driven by the D. melanogaster Actin 5c (Actin 5C) or three copies of the Pax3 (3xP3) promoters. The anti-Plasmodium engineered transgenes are composed of a secretory signal peptide derived from the Ae. aegypti Mal 1 gene13 cloned in-frame with the N2 single-chain antibody coding sequence and followed by an epitope tag (Etag). This construction is represented in the drawing by N2. Either the 3'UTR of an Ae. aegypti defensin gene (DF) or the SV40 polyadenylation signal (SV) abut the N2 open reading frame. Promoters of the Ae. aegypti Vitellogenin-1 gene (Vg1) and D. melanogaster Ubiquitin promoter (Ub) were cloned immediately 5' upstream of the N2 cassette.

 
pM[Vg-mal I-N2-Etag-SV40] (abbreviated to VgN2SV), was generated by NdeI restriction enzyme digestion of pGEM-T[mal I-N2-Etag-SV40],5 and the ends were blunted by fill-in with the Klenow fragment. After this, the Mal 1-N2-Etag-SV40 fragment was removed from the plasmid with SacII digestion and ligated into pBSKII(+) (Stratagene, La Jolla, CA) to generate pBSK[mal I-N2-Etag-SV40]. The SmaI-SacII DNA fragment containing the Ae. aegypti Vg1 gene promoter9 was excised from BSK [Vg] (gift of A. Raikhel and V. Kokoza, University of California, Riverside, CA) and ligated into the SpeI (blunted by fill-in) and SacII sites of pBSK[mal I-N2-Etag-SV40], generating pBSK [Vg-mal I-N2-Etag-SV40]. The Vg-mal I-N2- Etag-SV40 fragment contained in pBSKII(+) was excised and ligated into the EcoRI site of pM[Actin 5C- EGFP] (gift of P. Atkinson, University of California, Riverside) generating pM[Vg-mal I-N2-Etag-SV40] . pM[Vg-mal I-N2-Etag-Df] (abbreviated to VgN2DF) was generated by amplifying a 286-bp defensinA12 3'UTR fragment with the primers DefF-SalI (5'-GTCGACCGAAAGGACCAAC-3') and DefR-XhoI (5'-CTCGAGACCATTTAACA-3'). The template was pGEMT-defensin (gift of A. Raikhel and V. Kokoza, University of California, Riverside) and the amplicon ligated into pGEM-T plasmid (Promega, Madison, WI). The Mal 1-N2-Etag cassette was excised from pBSK[mal 1-N2-Etag-SV40] by XbaI restriction endonuclease digestion. The resulting DNA fragment was blunted by Klenow and ligated into the pBSK [Vg] SmaI site generating pBSK [Vg-mal I-N2-Etag]. The 3'UTR of the defensin gene was inserted into the double-digested SalI-XhoI sites of pBSK [Vg-mal I-N2-Etag]. The resulting [Vg- mal I-N2- Etag-3'UTRdef] fragment was excised from the plasmid by digestion with BssHII restriction and cloned into the AscI site of pMos[3xP3-EGFP], producing pM[Vg-mal I-N2-Etag-Def].

pH[Ub-mal I-N2-Etag-SV40] (abbreviated to UbN2SV) was made by moving the Mal1-N2-Etag-SV40 fragment from pGEM-T[mal I-N2-Etag-SV40] into pslfa1180,14 using SacII-XhoI enzymes generating the pslfa1180 [malI-N2-Etag-SV40]. The D. melanogaster Ub promoter was excised from pBC KS [Ub] (11; Z. Adelman, unpublished data) with SacII and NheI enzymes and ligated into pslfa1180 [malI-N2-Etag-SV40] using the corresponding sites to generate pslfa1180 fa [Ub-malI-N2-Etag-SV40]. The cassette [Ub-malI-N2-Etag-SV40] was released from pslfa1180 fa [Ub-malI-N2-Etag-SV40] by AscI restriction digestion and ligated into pH[3xP3-EGFPPaf]14 to generate pH[Ub-mal I-N2-Etag-SV40].

Generation of transgenic mosquitoes. Embryo microinjections were performed as described in Jasinskiene and others.15 The helper plasmids used were pKhsp82MOS16 and pHSHH1.9.17 Donor (transformation vectors) and helper plasmids were co-injected into embryos at concentrations of 0.5 and 0.3 µg/µL, respectively. After injection, embryos were heat-shocked 24 hours later at 40°C for 60 min to stimulate the production of transposase encoded by the helper plasmids. Injected embryos were kept for 5 days at 27°C, 80% humidity, before hatching. Surviving adult males, designated G0, were mated individually to females of the host strain to produce single-founder families. G0 females were mated in groups of 10–20 to host-strain males to produce pools. The resulting G1 progeny were screened as larvae for the expression of the transformation marker phenotype, EGFP, using a Leica (Wetzler, Germany) MZ12 GFP Plus or Zeiss M2 BIO fluorescence microscope. Pupae were sexed, and males and females were separated to insure female virginity in all experimental crosses.

RNA isolation and reverse transcription-polymerase chain reaction. Total RNA was extracted from two to three adult mosquitoes at 24 hours after blood meal (hPBM) or unfed females using Trizol (Invitrogen, Carlsbad, CA). RNA samples were incubated for 20 minutes at 37°C with RQ1 DNase (Promega, Madison, WI). The Qiagen One-Step reverse transcription-polymerase chain reaction (RT-PCR) kit was used to detect the presence of N2scFV and actin mRNA. Gene specific primers used for RT-PCRs were N2scFV-Mal3 (5'-GGACTAACCACCGGGTTGGACTGGTGGGAAGC-3') and N2scFV-Estop (5'-GCTCTAGACTATGCGGCACGCGG-3'), and for actin were forward (5'-AAGGCCAACCGTGAGAAGATGACT-3') and reverse (5'-GCTCGTTGCCAATGGTGATAC-3').

Mosquito infection assays. Transgenic and control (non-transformed) mosquitoes were allowed to feed in parallel for 15 minutes on P. gallinaceum–infected chickens (parasitemia 10–15%). Females fed to repletion were transferred to a separate cage and analyzed further. These procedures result routinely in 100% prevalence of mosquito infection. Midguts were dissected from a cohort of mosquitoes at 8–9 days after the infectious blood meal and evaluated with light microscopy to determine the prevalence and mean intensity of infection of oocysts. Salivary glands were dissected from the remaining mosquitoes with the Ub promoter-driven scFv on Day 13–15 after infection, and the tissues were evaluated for parasite presence and number. Mosquitoes with Vg1-driven scFvs were fed a second time on uninfected chickens at a time estimated to be 24–48 hours previous to oocyst rupture and release of sporozoites into the hemolymph. This estimate was based on the degree of development of the oocysts observed at Days 8–9 after infection. Fully engorged females were transferred to a separate cage, and their salivary glands were dissected and analyzed 13–15 days after the initial infection. Salivary gland pairs were dissected from individual mosquitoes and homogenized in 10 µL of phosphate-buffered saline (PBS). The complete homogenate was placed on a hemacytometer, and sporozoites were counted using phase-contrast microscopy. All fields of the hemacytometer were examined thoroughly. Five fields were counted and averaged in samples having high parasite densities, and all fields were counted in samples with low parasite densities.

Chicken infection assays. Experiments were conducted to determine the transmission capabilities of the transgenic and control mosquitoes. Single infected mosquitoes were allowed to feed on individual uninfected chickens (chickens used were either 7–14 or 21 days after hatching). After feeding, salivary glands were dissected, and the number of sporozoites was counted. Chickens were tested for infection beginning 7 days after exposure to parasite-infected mosquitoes. Erythrocytes were examined microscopically each day until a positive parasitemia was observed. Uninfected chickens were defined as those not having parasites at 21 days after the bites of infected mosquitoes.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 3,126 Ae. aegypti embryos were injected with the transformation vectors, and of these, 1,286 survived to produce G0 adults. Screening of the progeny of the fertile adults resulted in 23 transgenic male-founder families and three pools, yielding an average transformation frequency of 5.2% (range, 2.6–7.4%; Table 1Go).


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TABLE 1
Embryo microinjection and transformation results
 
Two lines each representing mosquitoes carrying the transgenes VgN2SV (lines 21 and 166), VgN2DF (lines 48 and 75), or UbN2SV (lines 131 and 256) were selected for further analyses. Southern blots performed with genomic DNA from these lines using radio-labeled probes containing sequences complementary to N2scFv or to the marker gene, EGFP, validated the integration of single copies of the transgene into the genome of each line (results not shown).

Gene amplification analyses (RT-PCR) were done to evaluate the expression of the transgenes in blood-fed mosquitoes. Diagnostic amplification products representative of N2scFv mRNA were detected in all tested transgenic lines (Figure 2Go). Although the results are not quantitative, no differences were seen in signal intensities when comparing lines with SV40 3'-end UTRs with those containing the DefA 3'-end UTR. No amplification products were obtained using mRNA derived from the respective untransformed control Higgs or RED strains.


Figure 2
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  		FIGURE 2. Gene amplification analyses of the transgene expression. Single-chain antibody (N2scFv) and control (Actin) gene transcription products were detected in samples of total RNA extracted from blood-fed mosquitoes analyzed by RT-PCR with specific primers for each gene. N2scFv mRNA was found in all six transgenic lines (21, 166, 48, 75, 131, and 256) and absent from control females from Higgs (H) and RED (R) strains. The transgene target products (VgN2SV, VgN2DF, and UbN2SV) are indicated at the top of each figure.

 
Control Higgs and RED mosquitoes fed on infected chickens had 100% prevalence of oocyst and sporozoite infections within 2 weeks of the blood meal (Table 2Go; Figure 3Go), and this is consistent with their high susceptibility to the avian parasite. Although oocyst numbers were variable, control mosquitoes developed high mean intensities of sporozoite infection (6,000 to ~17,000 sporozoites per salivary gland pair), with individual females having infections that ranged from 2,500 to > 28,000 parasites. Transgenic mosquitoes fed in parallel on the same infected chickens also had 100% prevalence of oocyst infections. Sporozoite prevalence was reduced (62–96%) in Experiments 3–8; however, it is not possible to attach statistical significance to this reduction, and the majority (105/ 119) of transgenic mosquitoes had salivary glands containing some sporozoites. Mean intensities of sporozoite infections and SDs were determined for each experimental group. However, SDs in some of the experiments exceeded the mean, indicating that our sample populations were not uniform. This was validated when we plotted the individual sporozoites numbers and indicates that we have two groups of mosquitoes: those in which we see an effect and those that are not affected (Figure 3Go). Approximately 40% (44/119) of the challenged transgenic mosquitoes had ≥ 5,000 sporozoites in their glands, numbers similar to those of non-transgenic mosquitoes. However, 34% of them (41/119) showed remarkably low numbers of parasites (< 100), and an additional 12% (14) had no detectable sporozoites in their glands. Mosquitoes with these low infection numbers were never seen in the controls. Reductions in mean intensities of infection varied from 52% to 90% in mosquitoes transformed with Vg1-driven N2scFvs (Table 2Go; Experiments 1–6), whereas the two Ub-driven lines showed a reduction of 62% (line 131) and essentially no reduction (line 256; Table 2Go; Experiments 7 and 8). No effects on prevalence or mean intensities of infection were evident among the lines differing in their 3'-end UTRs (Table 2Go; Experiments 1–4, 7, and 8 compared with Experiments 5 and 6). Lines with N2scFv driven by the Vg1 promoter produced a greater proportion (12/77 [~16%]; Figure 3Go) of mosquitoes with no parasites in their salivary glands in comparison with those with Ub-driven constructs (2/42 [~5%]; Figure 3Go).


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TABLE 2
Prevalence and mean intensities of infection of salivary-gland sporozoites in transformed and control mosquitoes
 

Figure 3
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  		FIGURE 3. Sporozoite infections in the salivary glands of individual transgenic and control Ae. aegypti. Salivary glands were dissected from transgenic (lines 48, 75, 21, 166, 131, and 256) and control (Higgs or RED) mosquitoes after feeding on P. gallinaceum–infected chickens, and the sporozoites counted using phase-contrast microscopy. Control mosquitoes were non-transgenic Ae. aegypti fed in parallel on the same infected chickens. Wide panels, number of sporozoite per salivary gland pair for all mosquitoes analyzed. Narrow panels, all experimental animals with sporozoite numbers below 70 per salivary gland pair. Solid circles represent individual mosquitoes with at least one sporozoite detected in their salivary glands, and open circles are mosquitoes with no sporozoites. Vg and Ub refer to the Vg1 and Ub-driven transgene constructs, respectively.

 
Chickens were challenged with bites of 13 individual infected transgenic mosquitoes from lines 21 and 166 that harbored a wide range of sporozoites in their salivary glands (Table 3Go). All infected mosquitoes (11/13), including those with parasite numbers as low as 20, were able to transmit malaria to their vertebrate hosts. The two mosquitoes with no detectable sporozoites failed to infect chickens.


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TABLE 3
Transmission blocking in transgenic mosquitoes
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Engineered antibodies with optimized functional properties are valuable research tools, and some have resulted in significant improvements of treatment of several human diseases.18,19 A number of antibody-based therapies have been proposed for the treatment and prevention of malaria.20 These include the development of recombinant scFvs containing parasite-specific binding domains derived from anti-Plasmodium monoclonal antibodies to act as blockers of indispensable receptor–ligand interactions between host cells and parasites, to function as opsonins, to activate vertebrate immunity or to be adapted as parasite-directed drug-delivering agents. Although these molecules could target parasites during their development in vertebrate hosts, they also are useful as antiparasite effector molecules in the vectors.5,21,22

Here we show that a single-chain antibody (N2scFv) expressed in transgenic mosquitoes can block parasite infection of salivary glands; this supports the hypothesis that scFvs can be used as anti-sporozoite effector molecules. A number of anti-Plasmodium effector molecules, including other scFvs, the SM1 peptide, and a honeybee phospholipase, have been validated in animal models of malaria transmission.3 These molecules reduce the infectivity of P. gallinaceum to Ae. aegypti and P. berghei to An. stephensi by blocking parasite development at the midgut stages (ookinete-oocysts).21–24 Furthermore, SM1 also blocks sporozoite invasion of salivary glands, but it is not straightforward to determine how much of the reduced sporozoite phenotype is caused by fewer oocysts. N2sFV therefore adds to the list of effector molecules to target Plasmodium in the mosquitoes at a different site and developmental stage (hemolymph sporozoites).

Two alternative cis-regulatory sequences (promoters), derived from the Ae. aegypti Vg1 gene and the D. melanogaster Ub gene, were tested. Both promoters have been validated in previous mosquito transformation studies and provide robust expression of transgenes under their transcriptional regulation.9,11 These regulatory sequences were selected based on the fact that the Ub promoter is expressed constitutively providing a continuous presence of the transgene product, whereas the Vg1 promoter is activated after a blood meal, offering the opportunity to induce transcription of the transgene only at a desired time, when sporozoites are present in the insect hemolymph. However, the proportion of mosquitoes with no or reduced sporozoites was consistently higher in mosquitoes carrying the transgenes driven by the Vg1 promoter than those with the Ub promoter. We interpret this to indicate that the concentration of N2scFv in the hemolymph provided by the Ub promoter is not as high as that attained with the Vg1 regulatory sequence. Therefore, under the experimental conditions applied in this study, the Vg promoter is more effective in driving expression of N2sFv to interfere with parasite development. Vitellogenin-encoding genes are among the most highly induced genes in the mosquito after a blood meal,25,26 and their expression in the fat body, a tissue that is functionally equipped for secretion of large amounts of protein within a short time into the hemolymph, may permit greater access of the effector molecule to the target parasites.

The variable effects on mean intensities of infection seen in the Vg1 N2scFv experiments are likely to result from the feeding schedule, and therefore, induction of the transgene, and variability in parasite maturation time. Although we tried to anticipate this variability by looking at oocyst development, the successful maturation of one or two oocysts before the second blood meal could result in sporozoites escaping the N2scFv. This variability prevented a rigorous statistical analysis of the data derived from all of the mosquitoes. However, it is important to emphasize that mosquitoes with no or low (< 100) numbers of parasites were never found among the control mosquitoes. These observations support efforts to optimize further the expression of the scFvs. Improvements of this scFv-based approach include the selection of Vg gene promoters that do not cycle after the blood meal27 and engineering of 5' and or 3'UTRs sequences to enhance stability of the mRNA. In addition, optimization of the codons usage in the scFv ORF based on the mosquito not mouse codon use is likely to boost protein expression and stabilization of the scFv molecules by enhancing proteolytic cleavage resistance. Furthermore, the generation of molecular variants such as bivalent or multivalent scFv-carrying complementary binding domains to target various Plasmodium surface antigens concomitantly could enhance the resistance phenotype. Fusion constructs in which the scFv binding capacity would deliver anti-Plasmodium molecules to the surface of the parasites also are alternatives.21

We anticipated that there would be a threshold in mean intensity of infection for sporozoites in the salivary glands, and if we could achieve that threshold, transmission would be interrupted. This expectation was supported by the work of Ito and others,23 who showed that if mean intensities of P. berghei were less than ~400 sporozoites in the salivary glands, mosquitoes were incapable of infecting mice. We cannot determine in these experiments the actual number of parasites delivered by mosquitoes to their vertebrate hosts during blood meals. However, our data showed that mosquitoes with as few as 20 sporozoites remaining in their salivary glands after feeding were capable of infecting chickens with the prior blood meal. We understandably were eager to determine which of these animal models is prognostic for human parasites. Work by Ungureanu and others28 showed that 10 sporozoites of P. vivax were capable of establishing a malaria infection after needle inoculation. Given these data, we take the most stringent endpoint and set zero as our target for mean intensity of sporozoite infections in salivary glands. This is essentially equivalent to zero prevalence of infectious forms in the glands. Thus, these data support the conclusion that transgenic mosquitoes with a "no sporozoite" phenotype will be necessary to effectively impact malaria transmission on use of a population replacement strategy.

The development of anti-sporozoite effector genes is ongoing work. Combining these genes with others that target ookinetes and prevent oocyst formation in a multigenic approach should permit producing the phenotype of "no sporozoites" in the salivary glands. Furthermore, the use of multiple effector genes is likely necessary to avoid the selection of resistance to any one mechanism, preventing the breakdown of a control strategy based on genetics approaches. In addition, a balance among fitness effects, effectiveness of the molecule, ease of engineering of the phenotypes, and the mechanism for spreading the phenotypes through a population will dictate the practicality of any one strategy.29

Received December 12, 2006. Accepted for publication January 31, 2007.

Acknowledgments: The authors thank the members of our laboratory for discussions and Lynn M. Olson for help in preparing the manuscript.

Financial support: This work was supported by awards from the National Institutes of Health (AI29746 and AI44800) and the Burroughs-Wellcome Fund.

* Address correspondence to Anthony A. James, Department of Microbiology and Molecular Genetics, Department of Molecular Biology and Biochemistry, 3205 McGaugh Hall, University of California, Irvine CA 92697-3900. E-mail: aajames{at}uci.edu Back

Authors’ addresses: Nijole Jasinskiene, Judy Coleman, Aurora Ashikyan, Michael Salampessy, Osvaldo Marinotti, and Anthony A. James, 3205 McGaugh Hall, MB&B, University of California, Irvine, CA 92697-3900. E-mail: aajames{at}uci.edu.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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D. H. Sieglaff, W. A. Dunn, X. S. Xie, K. Megy, O. Marinotti, and A. A. James
Comparative genomics allows the discovery of cis-regulatory elements in mosquitoes
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