Open Access

Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2

  • Seth A Hoffman1, 2, 3,
  • Lakshminarayanan Aravind2 and
  • Soundarapandian Velmurugan1Email author
Parasites & Vectors20125:27

DOI: 10.1186/1756-3305-5-27

Received: 12 January 2012

Accepted: 6 February 2012

Published: 6 February 2012

Abstract

Background

New interventions are required to optimally and sustainably control the Anopheles sp. mosquitoes that transmit malaria and filariasis. The mosquito olfactory system is important in host seeking (transmission) and mate finding (reproduction). Understanding olfactory function could lead to development of control strategies based on repelling parasite-carrying mosquitoes or attracting them into a fatal trap.

Findings

Our initial focus is on odorant binding proteins with differential transcript accumulation between female and male mosquitoes. We report that the odorant binding protein, OBP2 (AGAP003306), had increased expression in the antennae of female vs. male Anopheles gambiae sensu stricto (G3 strain). The increased expression in antennae of females of this gene by quantitative RT-PCR was 4.2 to 32.3 fold in three independent biological replicates and two technical replicate experiments using A. gambiae from two different laboratories. OBP2 is a member of the vast OBP superfamily of insect odorant binding proteins and belongs to the predominantly dipteran clade that includes the Culex oviposition kairomone-binding OBP1. Phylogenetic analysis indicates that its orthologs are present across culicid mosquitoes and are likely to play a conserved role in recognizing a molecule that might be critical for female behavior.

Conclusions

OBP2 has increased mRNA transcript accumulation in the antennae of female as compared to male A. gambiae. This molecule and related molecules may play an important role in female mosquito feeding and breeding behavior. This finding may be a step toward providing a foundation for understanding mosquito olfactory requirements and developing control strategies based on reducing mosquito feeding and breeding success.

Keywords

Anopheles gambiae Odorant Binding Protein Antennae

Findings

Research hypothesis

Factors that influence mosquito fitness, especially host seeking and mate finding are complex and modulated by multiple cues, of which olfactory cues are most important [14]. Detection of odor molecules requires odorant binding proteins (OBPs) that are abundant in antennal chemosensilla [5, 6]. OBPs are low molecular weight soluble proteins that bind and transport odor molecules from sensillae to G-protein-coupled receptors in olfactory sensory neurons [6]. The finding of receptor AgamOBP1 binding to its ligand indole demonstrated the significance of OBPs in odor recognition [7]. Understanding olfactory function could lead to development of malaria control strategies based on repelling Plasmodium sp. carrying Anopheles mosquitoes or attracting them into a fatal trap. A first step is assessment of expression of olfactory system associated genes [710]. There is sexually dimorphic expression of OBPs in Anopheles mosquitoes and Drosophila melanogaster[1113]. We are focusing on identifying OBPs in antennae of Anopheles gambiae, because in Africa A, gambiae is the most important vector of Plasmodium falciparum[14], a major vector of Wuchereria bancrofti, which causes lymphatic filariasis [15], and a vector of O'nyong-nyong virus [16]. In this study, based on results of a screening microarray (unpublished) and previous microarray studies [9, 11], we hypothesized that the OBP, OBP2 (AGAP003306), would have increased transcript accumulation by quantitative reverse transcription PCR (qRT-PCR) in female as compared to male A. gambiae antennae.

Methods

Collection and Processing of RNA

We studied AGAP003306, which had 2 fold greater expression in RNA isolated from antennae of 4 day old A. gambiae (Keele strain from Johns Hopkins) females than males in a microarray experiment (unpublished). In another microarray study of RNA from antennae of 5-7 day old Pink-eye A. gambiae, expression of AGAP003306 (OBP2) was 1.4 times higher in females as compared to males, but no qRT-PCR was done [9]. In yet another microarray study of RNA isolated from 3 day old whole mosquitoes (Pink-eye strain A. gambiae) there was approximately 3 fold increased expression in females vs males [17].

In this study, A. gambiae sensu stricto (G3 strain) were from the same batch of eggs (each batch giving rise to a mosquito lot) and were raised to adulthood under standard insectary conditions, and fed ad libitum with 10% sugar water [18]. We studied the antennae under controlled conditions of age and exposure to food. Adults of both sexes were collected exactly 4 days after emergence. The mosquitoes were immobilized by exposure to -20°C for 15 minutes, males and females separated, and antennae removed by manual dissection over dry ice, placed into separate 1.5 mL centrifuge tubes and homogenized using a pestle, each in 300 μL of Trizol reagent (Invitrogen, CA). RNA was isolated following manufacturer's instructions and purified using RNeasy mini column (Qiagen). The RNA was then assessed for quality and quantity using NanoDrop (ND-1000). The mosquito antennae that generated the RNA for the qRT-PCR experiments were isolated in July 2009 (mosquitoes from the University of Maryland), and January 2010 and June 2010 (mosquitoes from the National Institutes of Health).

qRT-PCR Assay

As an endogenous control, and foundation for the qRT-PCR analysis, we used the S7 ribosomal RNA gene of A. gambiae[19]. As another control we analyzed AGAP009629, which did not have differential expression in antennae of females vs. males by microarray, but had increased expression in antennae of unfed vs. blood-fed females (unpublished).

The primer pairs synthesized and used (Table 1) were designed using Primer 3[20]. The QuantiTect SYBR Green RT-PCR kit (Qiagen) was used and reactions conducted in 96 well plates for 40 cycles (Applied Biosystems StepOnePlus™ System). Each reaction contained 12.5 μL Qiagen 2x Master Mix, 0.25 μL forward and reverse primers (0.5 μM final concentration), 0.25 μL RT Mix (containing reverse transcriptase), 10.75 μL DEPC water and 1.0 μL of RNA (0.05-1 μg/μL). The 96 well plate was sealed with adhesive film and centrifuged at 3700 rpm for 1 minute at 4°C. The ribosomal S7 gene was used as an endogenous control and a reaction without RT Mix (reverse transcriptase) was included for all reactions as a negative control. Changes in threshold cycles (ΔΔCT) analysis was done to assess the ratio of RNA expression in females vs. males using previously published methods for analysis (Step One Software, v2.2, Applied Biosystems [21, 22]).
Table 1

Primers used in qRT-PCR

AGAP ID

Forward primer

Reverse primer

AGAP003306

CTGCACATGGGCAAGCTG

CGTTCGCACACATCCTTG

AGAP009629

GCGCTCCCAAGTTCAAAGTA

CGCAATGCACATCGTGTAG

Ribosomal S7

TGCGGCTTCAGATCCGAGTTC

TTCGTTGTGAACCCAAATAAAAATC

Phylogenetic analysis

Multiple sequence alignments were built using the KALIGN program [23], followed by manual adjustments on the basis of profile-profile and structural alignments. Phylogenetic analysis was conducted using an approximately-maximum-likelihood method implemented in the FastTree 2.1 program under default parameters [24].

Results

qRT-PCR

RNA was extracted from paired antennae of approximately 100 female and 100 male A. gambiae derived from the same batch of eggs and raised in the same cage that had never been exposed to a blood meal. 80 ng RNA/mosquito to 560 ng RNA/mosquito (mean of 270 ng RNA/mosquito) was obtained. Approximately double the RNA was obtained from female as compared to male antennae. We isolated antennae on three separate occasions from three separate batches of mosquitoes over a period of 2 years. The results of three assays using RNA isolated from antennae (from three separate lots of mosquitoes) at three different times and normalized to the expression of the S7 ribosomal RNA gene are shown in Table 2. In all experiments there was an increase in accumulation of transcripts of AGAP003306 in female vs. male A. gambiae (5 to 25 fold). When these data were integrated (Figure 1), there was a statistically significant increase in transcript accumulation of AGAP003306 in females vs. males (p = 0.037, paired student's t test). Furthermore, there was no significant difference in transcript accumulation between females and males of the control gene, AGAP009629 (p = 0.246, paired student's t test).
Table 2

Expression of AGAP003306 (OBP2) and AGAP0099629 (control) relative to expression of S7 in antennae of female and male A. gambiae in three biological replicates (experiments 1, 2 and 3).

Target

Experiment

Female

Male

Relative Expression

  

Cт Mean

Cт SD

ΔCт Mean

Cт Mean

Cт SD

ΔCт Mean

ΔΔCт

RQ

 

1

35.55

0.105

 

36.51

0.257

   

Reference S7

2

33.31

0.335

 

35.04

0.125

   
 

3

33.57

0.565

 

32.05

0.188

   
 

1

29.77

0.208

-5.780

33.87

0.414

-2.634

-3.145

8.849

AGAP003306

2

29.77

0.168

-3.540

33.87

0.414

-1.170

-2.370

5.171

 

3

30.85

0.091

-2.721

33.99

0.309

1.941

-4.662

25.309

 

1

35.42

0.298

-0.130

36.06

0.353

-0.450

0.320

0.801

AGAP009629

2

35.55

0.105

2.239

36.56

0.390

1.518

0.721

0.607

 

3

36.46

0.478

2.894

34.95

0.686

2.894

0.000

1.000

= (cycle threshold) number of cycles required for the fluorescent signal to reach threshold

Cт Mean = Average Cт from triplicate values on same sample in same PCR reaction

Cт SD = Standard Deviation of triplicate Cт

ΔCт Mean = Cт MeanTarget- Cт MeanReference (target=AGAP003306 or AGAP009629; reference=S7)

ΔΔCт = ΔCт MeanFemale - ΔCт MeanMale

RQ = Relative Quantification = 2-(ΔΔCт)

https://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-27/MediaObjects/13071_2012_Article_519_Fig1_HTML.jpg
Figure 1

Comparative expression of AGAP003306 in female and male A. gambiae. Columns show mean of ΔCт Mean values (normalized amount of AGAP003306 RNA present) of the three experimental replicates shown in Table 1 multiplied by -1, error bars are standard errors. Data are significantly different in a pair student's t-test (p = 0.037, n = 3).

The relative increased accumulation of OBP2 transcripts in females vs. males in the three experimental (biological) replicates varied (Table 2). The mosquito antennae that generated the RNA for the qRT-PCR experiments were isolated in July 2009 and were from mosquitoes from the University of Maryland (experiment 1), and in January 2010 (experiment 2) and in June 2010 (experiment 3) both from the National Institutes of Health, Bethesda. Thus, the differences in relative transcript accumulation were likely due to biological variability in gene expression from mosquitoes from different laboratories studied at different times. To determine if this was the case rather than variability of the assay, we repeated the qRT-PCR assays using the RNA of experiments 2 and 3. These technical replicates showed similar results as the original experiments (Table 3). In a recent publication, similar results for the OBP2 gene were found in a single biological replicate of RNA from 4-6 day old A. gambiae by RNA-seq [13].
Table 3

Expression of AGAP003306 (OBP2) relative to expression of S7 in antennae of female and male A. gambiae in two technical replicates.

Target

Technical replicate

Female

Male

Relative Expression

  

Cт Mean

Cт SD

ΔCт Mean

Cт Mean

Cт SD

ΔCт Mean

ΔΔCт

RQ

 

Experiment 2

33.314

0.335

 

35.043

0.125

   

Reference

 

35.553

0.105

 

36.507

0.257

   

S7

Experiment 3

33.565

0.565

 

32.053

0.188

   
  

36.502

0.309

 

31.034

0.400

   
 

Experiment 2

29.774

0.168

-3.540

33.873

0.414

-1.170

-2.371

5.172

AGAP

 

31.725

0.376

-3.828

34.759

0.191

-1.749

-2.079

4.226

003306

Experiment 3

30.845

0.091

-2.721

33.994

0.309

1.941

-4.662

25.309

  

33.167

0.384

-3.335

32.711

0.562

1.678

-5.013

32.293

= (cycle threshold) number of cycles required for the fluorescent signal to reach threshold

Cт Mean = Average Cт from triplicate values on same sample in same PCR reaction

Cт SD = Standard Deviation of triplicate Cт

ΔCт Mean = Cт MeanTarget- Cт MeanReference (target=AGAP003306 or AGAP009629; reference=S7)

ΔΔCт = ΔCт MeanFemale - ΔCт MeanMale

RQ = Relative Quantification = 2-(ΔΔCт)

Phylogenetic Analysis

OBP2 belongs to an OBP super family that includes the insect pheromone binding proteins [6]. Another member of this family, Agam OBP1, mediates indole recognition in antennae of female A. gambiae[7]. The olfactory receptors of terrestrial animals exist in an aqueous environment; yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via OBPs, which exist in the extracellular fluid surrounding odorant receptors. This family includes proteins that specialize in binding insect pheromones (PBPs) and others that bind general odorants (GOBPs) [6]. Prior phylogenetic analysis has suggested that evolution of the OBP superfamily has evolved primarily through the process of lineage-specific expansion [25]. Thus, the majority of the OBPs in a given lineage such as Diptera, Hymenoptera, Lepidoptera or Coleoptera tend to cluster with others from the same lineage to the exclusion of those from other lineages. The genome of A. gambiae itself contains about 72 members of the OBP family.

We performed a phylogenetic analysis using over 100 representative OBPs from dipterans, hymenopterans and coleopterans with completely sequenced genomes. OBP2 is lodged within a predominantly dipteran lineage-specific expansion of OBPs that are particularly well represented in the culicid mosquitoes (Figure 2). This clade of OBPs includes the Culex (e.g. CquiOBP1) OBP that binds the oviposition kairomones (5R, 6S)-6-acetoxy-5-hexadecanolide [26] (marked red, Figure 2). This analysis also showed that orthologs of A. gambiae OBP2 are conserved across Culex, Aedes, and Anopheles genera but are absent in Drosophila (blue box, Figure 2), pointing to a function for this protein in potentially binding a conserved odorant molecule in culicid mosquitoes. The up regulation of OBP2 observed in females as compared to males, suggests it could possibly bind a molecule comparable to the oviposition kairomone bound by OBP1. However, such a kairomone could also have an alternative role in guiding female feeding behavior. On the other hand it is also possible that OBP2 binds a pheromone that males express. The focus of future studies would be to pinpoint the role of this protein by determining the impact of knocking it down vis-a-vis feeding behavior and fitness of females.
https://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-27/MediaObjects/13071_2012_Article_519_Fig2_HTML.jpg
Figure 2

Phylogenetic analysis. Phylogenetic analysis was done using over 100 representative OBPs from dipterans (magenta), hymenopterans (turquoise) and coleopterans (yellow). The light blue box shows that orthologs of A. gambiae OBP2 are conserved across Culex, Aedes, and Anopheles genera, but are absent in Drosophila. The orange box shows that this clade of OBPs includes the Culex (e.g. CquiOBP1) OBP that binds the oviposition kairomones (5R, 6S)-6-acetoxy-5-hexadecanolide. The lineage-specfic expansions in various insect lineages other than the OBP1 and OBP2 clades, which are discussed in the paper have been collapsed and the branch lengths equalized for simplicity of viewing.

New interventions are needed to control the mosquitoes that transmit the parasites that cause malaria [27, 28] and lymphatic filariasis. Despite exciting scientific advances during the past few decades, no new approaches to mosquito vector control have been translated into widely used effective interventions. Sequencing the A. gambiae genome [29] and transcriptomics have provided a foundation for an approach to developing new interventions based on identifying genes and gene products that are important in transmission and mate-seeking. Stable genetic knockouts have not been generated in A. gambiae. However, transient knockdown by injection of sRNAi can be done and used to confirm the functional importance of OBP2 and other genes. This will be one of the next steps in our work.

Declarations

Acknowledgements

We particularly appreciate Dr. Peter Billingsley's support and guidance with data analysis and illustration and with manuscript finalization. We are grateful to Dr. George Dimopoulos, Johns Hopkins School of Public Health for initiation of this project, guidance and support. We thank Yuemei Dong, and Antonio M. Mendes, Johns Hopkins School of Public Health, for support with our unpublished microarray studies. We thank Robert Harrel, Dr. David O'Brochta, and Robert Alford of the University of Maryland Biotechnology Institute and Dr. Tobi Lehmann from NIAID, for providing A. gambiae for the qRT-PCR studies. We thank Dr. Anthony James for review of the manuscript. We are indebted to the team at Sanaria Inc., especially Yonas Abebe, Solomon Conteh, Dr. Abraham Eappen, Dr. Adriana Ahumada, Dr. Anusha Gunasekera, and Benjamin Hoffman for logistical and technical support and discussions.

Authors’ Affiliations

(1)
Sanaria Inc., 9800 Medical Center Dr.
(2)
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health
(3)
Cornell University

References

  1. Zwiebel LJ, Takken W: Olfactory regulation of mosquito-host interactions. Insect Biochem Mol Biol. 2004, 34: 645-652. 10.1016/j.ibmb.2004.03.017.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Rutzler M, Zwiebel LJ: Molecular biology of insect olfaction: recent progress and conceptual models. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2005, 191: 777-790. 10.1007/s00359-005-0044-y.View ArticlePubMedGoogle Scholar
  3. Chen XG, Mathur G, James AA: Gene expression studies in mosquitoes. Adv Genet. 2008, 64: 19-50.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Maekawa E, Aonuma H, Nelson B, Yoshimura A, Tokunaga F, Fukumoto S, Kanuka H: The role of proboscis of the malaria vector mosquito Anopheles stephensi in host-seeking behavior. Parasit Vectors. 2011, 4: 10-10.1186/1756-3305-4-10.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Vogt RG, Riddiford LM: Pheromone binding and inactivation by moth antennae. Nature. 1981, 293: 161-163. 10.1038/293161a0.View ArticlePubMedGoogle Scholar
  6. Pelosi P, Zhou JJ, Ban LP, Calvello M: Soluble proteins in insect chemical communication. Cell Mol Life Sci. 2006, 63: 1658-1676. 10.1007/s00018-005-5607-0.View ArticlePubMedGoogle Scholar
  7. Biessmann H, Andronopoulou E, Biessmann MR, Douris V, Dimitratos SD, Eliopoulos E, Guerin PM, Iatrou K, Justice RW, Krober T: The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes. PLoS One. 2010, 5: e9471-10.1371/journal.pone.0009471.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Pitts RJ, Fox AN, Zwiebel LJ: A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae. Proc Natl Acad Sci USA. 2004, 101: 5058-5063. 10.1073/pnas.0308146101.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Biessmann H, Nguyen QK, Le D, Walter MF: Microarray-based survey of a subset of putative olfactory genes in the mosquito Anopheles gambiae. Insect Mol Biol. 2005, 14: 575-589. 10.1111/j.1365-2583.2005.00590.x.View ArticlePubMedGoogle Scholar
  10. Xu W, Cornel AJ, Leal WS: Odorant-binding proteins of the malaria mosquito Anopheles funestus sensu stricto. PLoS One. 2010, 5: e15403-10.1371/journal.pone.0015403.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Schymura D, Forstner M, Schultze A, Krober T, Swevers L, Iatrou K, Krieger J: Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae. Int J Biol Sci. 2010, 6: 614-626.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Zhou S, Stone EA, Mackay TF, Anholt RR: Plasticity of the chemoreceptor repertoire in Drosophila melanogaster. PLoS Genet. 2009, 5: e1000681-10.1371/journal.pgen.1000681.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Pitts RJ, Rinker DC, Jones PL, Rokas A, Zwiebel LJ: Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding. BMC Genomics. 2011, 12: 271-10.1186/1471-2164-12-271.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Himeidan YE, Elzaki MM, Kweka EJ, Ibrahim M, Elhassan IM: Pattern of malaria transmission along the Rahad River basin, Eastern Sudan. Parasit Vectors. 2011, 4: 109-10.1186/1756-3305-4-109.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Dunyo SK, Appawu M, Nkrumah FK, Baffoe-Wilmot A, Pedersen EM, Simonsen PE: Lymphatic filariasis on the coast of Ghana. Tran R Soc Trop Med Hyg. 1996, 90: 634-638. 10.1016/S0035-9203(96)90414-9.View ArticleGoogle Scholar
  16. Lanciotti RS, Ludwig ML, Rwaguma EB, Lutwama JJ, Kram TM, Karabatsos N, Cropp BC, Miller BR: Emergence of epidemic O'nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus. Virology. 1998, 252: 258-268. 10.1006/viro.1998.9437.View ArticlePubMedGoogle Scholar
  17. Marinotti O, Calvo E, Nguyen QK, Dissanayake S, Ribeiro JM, James AA: Genome-wide analysis of gene expression in adult Anopheles gambiae. Insect Mol Biol. 2006, 15: 1-12. 10.1111/j.1365-2583.2006.00610.x.View ArticlePubMedGoogle Scholar
  18. Benedict MQ: Care and maintenance of anopheline mosquito colonies. The Molecular Biology of Insect Disease Vectors: A Methods Manual. Edited by: Crampton JM, Beard CB, Louis C. 1997, London: Chapman and Hall, 1: 3-12.View ArticleGoogle Scholar
  19. Salazar CE, Mills-Hamm D, Kumar V, Collins FH: Sequence of a cDNA from the mosquito Anopheles gambiae encoding a homologue of human ribosomal protein S7. Nucleic Acids Res. 1993, 21: 4147-10.1093/nar/21.17.4147.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000, 132: 365-386.PubMedGoogle Scholar
  21. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
  22. Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.View ArticlePubMedGoogle Scholar
  23. Lassmann T, Frings O, Sonnhammer EL: Kalign2: high-performance multiple alignment of protein and nucleotide sequences allowing external features. Nucleic Acids Res. 2009, 37: 858-865. 10.1093/nar/gkn1006.PubMed CentralView ArticlePubMedGoogle Scholar
  24. Price MN, Dehal PS, Arkin AP: FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010, 5: e9490-10.1371/journal.pone.0009490.PubMed CentralView ArticlePubMedGoogle Scholar
  25. Lespinet O, Wolf YI, Koonin EV, Aravind L: The role of lineage-specific gene family expansion in the evolution of eukaryotes. Genome Res. 2002, 12: 1048-1059. 10.1101/gr.174302.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Mao Y, Xu X, Xu W, Ishida Y, Leal WS, Ames JB, Clardy J: Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone. Proc Natl Acad Sci USA. 2010, 107: 19102-19107. 10.1073/pnas.1012274107.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Breman JG, Alilio MS, Mills A: Conquering the intolerable burden of malaria: what's new, what's needed: a summary. Am J Trop Med Hyg. 2004, 71: 1-15.PubMedGoogle Scholar
  28. WHO: The global malaria action plan for a malaria free world. Geneva: Roll Back Malaria Partnership (RBMP). WHO. 2008, 274:Google Scholar
  29. Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JM, Wides R: The genome sequence of the malaria mosquito Anopheles gambiae. Science. 2002, 298: 129-149. 10.1126/science.1076181.View ArticlePubMedGoogle Scholar

Copyright

© Hoffman et al; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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