Vanderbilt University
VICB Logo

home research discoveries core facilities training & research opportunities seminars & events news giving contact

 

Discoveries Featured

Linking the Sense of Smell to Reproduction in Disease Vector Mosquitoes

By: Carol Rouzer, Marnett Research Laboratory
Published: February 10, 2014


Odorant receptors expressed in mosquito sperm modulate sperm motility.

Malaria remains a preeminent public health problem in the developing world, with 219 million cases in 2010, resulting in 660,000 deaths, 91% of which were in Africa.  The cause of malaria is a parasite carried by approximately 40 species of Anopheline mosquitoes (Figure 1).  Extensive efforts to develop new drugs to treat or a vaccine to prevent the disease have met with limited success, suggesting that the best way to defeat malaria may be to control the mosquito vector. This has led Vanderbilt Institute of Chemical Biology member Laurence Zwiebel and his laboratory to seek biochemical vulnerabilities in mosquito physiology that may be exploited to prevent mosquito feeding, reproduction, or survival. Now, they report that proteins that enable the female mosquito to find a blood meal host also play a role in modulating the motility of mosquito spermatozoa [R. J. Pitts et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online February 3, doi:10.1073.pnas.1322923111].

      

Figure 1
. Anopheles freeborni mosquito taking a blood meal. Image reproduced from the Centers for Disease Control http://www.cdc.gov/malaria/about/biology/mosquitoes, CDC, public domain.


Mosquitoes of the species Anopheles gambiae are the most important malaria vectors.  There are five chemosensory gene families in these mosquitoes, including odorant receptors (AgOr), gustatory receptors (AgGr), ionotropic glutamate receptors (AgIr), and odorant binding proteins (AgObp).  The odorant receptors, which play an important role in blood meal-seeking behaviors, are heteromeric ligand-gated ion channels comprising at least two subunits, an AgOr odorant (ligand)-binding receptor protein, which can be viewed as a “tuning Or”, and the odorant receptor coreceptor protein (AgOrco). The AgOr protein establishes the specificity of the odorant receptor, and the 75 different AgOr genes in the An. gambiae genome provide the insect with the ability to detect a diverse array of chemicals. The wide range of AgOr genes expressed in the mosquito antennae are critical to feeding behavior. However, AgOr genes are also expressed in other tissues, where their function is unknown. Indeed, data from RNA sequence (RNAseq) studies carried out in 2010 revealed a subset of AgOr genes that was more highly expressed in the bodies of male than female mosquitoes. In light of several studies suggesting that mammalian odorant receptors, which act as G-protein coupled receptors, play a role in directed motility in sperm, the Zwiebel lab hypothesized their counterparts may function similarly in the mosquito.

In support of this hypothesis, specific RNA sequence (RNAseq) studies of the testis demonstrated expression of more than 30 AgOr genes, many of which were expressed more highly in the testis than in the antennae. These results suggested that odorant receptors may play a role in spermatogenesis or sperm function, although, surprisingly, attempts to detect transcripts of AgOrco were unsuccessful. Thus, it appeared that the gene for the key odorant receptor partner protein was not actively expressed in the testis.

The expression of multiple AgOr genes in the absence of AgOrco was unexpected, leading the researchers to postulate that AgOrco might be expressed very early in spermatogenesis, producing a stable protein that did not require continual synthesis. They first used Multidimensional Protein Identification Technology (MudPIT) to search for the AgOrco protein in mosquito testis tissue, but they found that this approach lacked sufficient sensitivity. They obtained better results with an antibody directed against the Orco protein, which revealed the presence of AgOrco in multiple developmental zones of the An. gambiae testis. Although AgOrco levels were highest in the regions corresponding to early spermatogenesis, the protein could also be seen in the flagella of mature spermatozoa, where it co-localized with α-tubulin (Figure 2).

Figure 2. Expression of AgOrco protein in the testes of Anopheles gambiae mosquitoes. (A) A differential contrast image of a whole mosquito testis shows the different zones of spermatogenesis demarcated with dotted lines. (B) The presence of the AgOrco protein in whole testis is revealed by immunofluorescence with the use of an anti-Orco antibody (green). Cell nuclei are counterstained with propidium iodide (magenta). The inset shows an individual spermatozoan, with green staining indicating the presence of AgOrco in the flagellum (f), but not in the midpiece (m) or head (h). (C) Close-up view of AgOrco immunofluorescence staining in the germ cell/spermatogonia region of the testis (outlined with a dotted line in (B)). Image reproduced with permission from J. R. Pitts, et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online February 3, doi:10.1073.pnas.1322923111, copyright 2014, J. R. Pitts, et al.

 

The presence of both tuning AgOrs and AgOrco suggested that functional odorant receptors could play a role in either spermatogenesis or sperm function. To test the hypothesis that odorant receptors modulate sperm motility, the investigators developed a video-based bioassay to quantify the motion of sperm flagellae (Figure 3). They placed a mosquito testis between a microscope slide and a coverslip and gently disrupted the tissue to release intact spermatozoa; test compounds could then be applied at varying concentrations to the fluid bathing the testis. A video camera recorded the motility of the sperm escaping from the testis. Blinded observers then viewed the recordings and rated the motion of the sperm flagellae on a scale from zero to three.



Figure 3.
  Figure 3. Design of the sperm motility assay. A mosquito testis is immersed in buffer containing test compound between a microscope slide and a coverslip. A videocamera records the motility (beating of the flagella) of sperm that escape from the disrupted testis. Motility is rated by a blinded observer on a scale from zero to three. Image reproduced with permission from J. R. Pitts, et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online February 3, doi:10.1073.pnas.1322923111, copyright 2014, J. R. Pitts, et al.


Using their motility assay, the researchers found that two Orco allosteric agonists (VUAA1 and VUAA4), discovered at the VICB, stimulated sperm motility, while an inactive analog (VUAA0) and an antagonist (VUANT) had no effect when added alone to the preparation. VUANT blocked the activity of VUAA1 and VUAA4. These findings supported a role for AgOrco in promoting sperm motility. In addition, fenchone, a ligand for AgOr11, and indole-3-carboxaldehyde, a ligand for AgOr6, stimulated beating of the sperm flagellae. These responses were also blocked by VUANT, supporting a role for AgOr proteins in AgOrco-mediated sperm activation. Motility was also stimulated by 8-Br-cAMP, a membrane-penetrant analog of cAMP. This effect was not blocked by VUANT, suggesting that cAMP acts as a second messenger in sperm activation independently of odorant receptor activation.

The investigators extended their studies to Aedes aegypti mosquitoes, which are important vectors for dengue, yellow fever, and chikungunya viruses, where Orco-/-, mutants were recently generated. In these important control experiments, Zwiebel lab researchers observed that spermatozoa from Orco-/- mutant insects did not respond to VUAA4, while wild-type spermatozoa did. Both mutant and wild-type spermatozoa responded to 8-Br-cAMP. Attempts to perform similar experiments using sperm from Orco-/- and wild-type fruit flies (Drosophila melanogaster) were hampered by a high background of sperm motility in the absence of any activating compound. Furthermore, the researchers confirmed the presence of Orco protein in the spermatozoa of wild-type D. melanogaster, the parasitic wasp Nasonia vitripennis, and the mosquito Aedes albopictus, suggesting that odorant receptors may play a role in sperm function in many insect species (Figure 4).

Figure 4. Expression of Orco in sperm from various insect species. In each case, Orco is stained in green, α-tubulin (flagella) in blue, and nuclei in magenta.  Sperm from (A) wild-type D. melanogaster (B) Orco-/- D. melanogaster (C) N. vitripennis, (D) N. vitripennis pretreated with an Orco blocking peptide (E) Ae. albopictus and (F) Ae. albopictus pretreated with an Orco blocking peptide are shown. Image reproduced with permission from J. R. Pitts, et al. (2014) Proc. Natl. Acad. Sci. U.S.A., published online February 3, doi:10.1073.pnas.1322923111, copyright 2014, J. R. Pitts, et al.

The investigators note that, as is the case for many insects, female A. gambiae mosquitoes usually mate only once, and they then store the sperm for an extended period of time in their spermatheca. Thus, it is possible that the female produces substances that act on sperm odorant receptors to stimulate and direct sperm motility. Support for this hypothesis comes from prior work in D. melanogaster, showing that ablation of spermatheca secretory cells results in failure to fully activate spermatozoa and a loss of fertility in the female. Further work will be needed to fully elucidate the role of odorant receptors in mosquito sperm function; however this work may provide important clues to new ways to interfere with mosquito reproduction, leading to better methods of mosquito - and thereby malaria and other mosquito-borne disease - control.

 

 

 

 

 

 


 

 
     

   vicb_youtube_channel_mark


Vanderbilt University School of Medicine | Vanderbilt University Medical Center | Vanderbilt University | Eskind Biomedical Library

The Vanderbilt Institute of Chemical Biology 896 Preston Building, Nashville, TN 37232-6304 866.303 VICB (8422) fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2013 by Vanderbilt University Medical Center