Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2
© Hoffman et al; licensee BioMed Central Ltd. 2012
Received: 12 January 2012
Accepted: 6 February 2012
Published: 6 February 2012
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.
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.
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.
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 [1–4]. 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 . The finding of receptor AgamOBP1 binding to its ligand indole demonstrated the significance of OBPs in odor recognition . 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 [7–10]. There is sexually dimorphic expression of OBPs in Anopheles mosquitoes and Drosophila melanogaster[11–13]. We are focusing on identifying OBPs in antennae of Anopheles gambiae, because in Africa A, gambiae is the most important vector of Plasmodium falciparum, a major vector of Wuchereria bancrofti, which causes lymphatic filariasis , and a vector of O'nyong-nyong virus . 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.
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 . 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 .
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 . 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).
As an endogenous control, and foundation for the qRT-PCR analysis, we used the S7 ribosomal RNA gene of A. gambiae. 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).
Primers used in qRT-PCR
Multiple sequence alignments were built using the KALIGN program , 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 .
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).
Expression of AGAP003306 (OBP2) relative to expression of S7 in antennae of female and male A. gambiae in two technical replicates.
OBP2 belongs to an OBP super family that includes the insect pheromone binding proteins . Another member of this family, Agam OBP1, mediates indole recognition in antennae of female A. gambiae. 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) . Prior phylogenetic analysis has suggested that evolution of the OBP superfamily has evolved primarily through the process of lineage-specific expansion . 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.
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  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.
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.
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