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A spatial map of antennal-expressed olfactory ionotropic receptors in the malaria mosquito

Joshua I. Raji, Christopher J. Potter

Preprint posted on May 12, 2022 https://www.biorxiv.org/content/10.1101/2022.05.11.491386v1

Would an olfactory receptor in any other place smell just as sweet? Ionotropic receptor position on the mosquito antenna.

Selected by T. W. Schwanitz

Background: Location, location, location: as with real estate, location matters for olfactory receptors. Mosquito olfactory receptors are mostly located on the antennae (Fig. 1A). But how are olfactory receptors distributed there, and how does that affect their sense of smell? In Drosophila melanogaster, the structures that house olfactory receptors—the sensilla—are localized to specific regions of the antennae (Vosshall and Stocker, 2007). In contrast, mosquito antennae have a more regular distribution of sensilla (Seenivasagan et al., 2009). Nevertheless, when Raji and Potter investigated the spatial distribution of the ionotropic receptors housed within these sensilla, they found that the receptors were not evenly distributed.

To detect airborne molecules, the mosquito olfactory system uses many different types of receptors, such as gustatory receptors, odorant receptors, and ionotropic receptors. The latter are ion channels that have a binding site for specific odorants. Usually, a “tuning ionotropic receptor” is tuned to a certain odorant or class of odorants. It forms a complex with an “ionotropic coreceptor,” an ionotropic receptor that isn’t linked to just one group of compounds. Ionotropic receptors appear to be especially good at detecting acids and amines; hence, they may influence the preference some mosquitoes have for biting human beings, as amines could be one component of human odor that attracts mosquitoes (Chen et al., 2019).

The two species investigated in this preprint are both members of the confusing Anopheles gambiae species complex: An. gambiae s. s. (S form) and An. coluzzii (M form). While the exact taxonomic classification of these mosquitoes is unclear, their effect on humanity is hard to overlook—the malaria parasite that they transmit has been and still is one of the deadliest human pathogens. They make such good vectors because of their predilection for biting human beings and their preference for human odor. Deeper understanding of mosquito ionotropic receptors could facilitate better mosquito control methods.

 

Fig. 1A. Head of the Anopheles gambiae mosquito, with a schematic to show the antennae and mouthparts.

 

Key findings:

  1. Using whole-mount fluorescent in situ hybridization for four tuning ionotropic receptors (IRs 7w, 41c, 41t.2, 75l) and three ionotropic coreceptors (IRs 25a, 76b, and 8a), the authors show that these receptors are not evenly distributed on the antenna. Generally, tuning ionotropic receptors tend to be on the part of the antenna furthest from the head. Even two of the three coreceptors tested (IRs 25a and 76b) were most expressed on the distal half of the antenna (while IR8a seems to be common throughout the antenna; see Fig. 2).
  2. These results were consistent across multiple individuals, though there are notable differences between males and females, e.g., males do not express IRs on the first ten segments closest to their head and IR41t.2 expression is enriched in male antennae.
  3. The authors conducted a transcriptome analysis and then used that data to conduct in situ probes of all 20 additional ionotropic receptors that they found upregulated in the transcriptome. Again, they revealed that these receptors tend to be most expressed toward the distal end of the antenna. IR93a makes for a striking example, as 80% of the cells expressing it were on the 13th flagellomere, which is the final antennal segment at the very tip. Testing a limited number of these receptors in males confirmed the trend observed in females.
  4. Next, Raji and Potter used two probe whole mount in situs to examine co-expression among ionotropic coreceptors and four highly expressed tuning receptors. They found numerous interesting trends, e.g., IRs 7w and 75l overlap with IR8a, a coreceptor that has been previously linked to acid-sensitive tuning ionotropic receptors. This overlap suggests that the two tuning receptors could also be linked to acids. These results show that simply examining patterns of intersection can lead to hypotheses about receptor function.
  5. The authors also found that the number of ionotropic receptors expressed per flagellomere (antennal segment) seems to be consistent across individuals; however, the exact location of a receptor within a flagellomere is highly variable among individuals.

 

Fig. 2. Different receptors fluorescing along the mosquito antennae. Next to each full antenna is an inset of the region with the highest expression. Note that in D, IR751 is most expressed toward the proximal part of the antenna while the other receptors shown are most expressed in the distal portion.

 

Questions for the authors:

  1. You mention that your study is a “snapshot” of the expression of ionotropic receptors in mosquitoes that are five-to-seven days old. Are there any plans to look at other age ranges? Could there be any interesting insights from looking at ionotropic receptor expression in larvae or pupae? And would your in situ probes be possible in larval antennae?
  2. There are a lot of tantalizing instances of co-expression in your paper that seem like they could be worth follow-up studies, e.g., IR751 and IR8a being co-expressed suggests that IR751 is likely sensitive to acids. If you could only do functional studies on one of these combinations (empty neuron studies, for example), which combination would you investigate further, and why?
  3. Any speculation as to why ionotropic receptors are most expressed on the distal portion of the antenna and not the proximal portion?
  4. Do you think that odorant receptors could share a similar pattern of distribution along the antenna?

Why I think this study is important:

Where is it? What is it? How does it work? These are some of the most fundamental questions that can be asked about something, and they serve as the basis for more complex follow-up questions. By shedding light (and fluorescence) on the location of ionotropic receptors, Raji and Potter lay the groundwork for more precise studies. Knowing that a given receptor is mostly found on the distal part of the antenna means that studies investigating this receptor could focus on collecting only that part of the antennae, thus reducing background noise. Moreover, the authors make use of an interesting method that can reveal new insights about the function of different receptors.

References:

Chen, Z., Liu, F., & Liu, N. (2019). Human odour coding in the yellow fever mosquito, Aedes aegypti. Scientific reports9(1), 1-12.

Seenivasagan, T., Sharma, K. R., Shrivastava, A., Parashar, B. D., Pant, S. C., & Prakash, S. (2009). Surface morphology and morphometric analysis of sensilla of Asian tiger mosquito, Aedes albopictus (Skuse): an SEM investigation. Journal of vector borne diseases46(2), 125.

Vosshall, L. B., & Stocker, R. F. (2007). Molecular architecture of smell and taste in Drosophila. Annu. Rev. Neurosci.30, 505-533.

Tags: insects, irs, mosquitoes, olfaction

Posted on: 2nd June 2022

doi: https://doi.org/10.1242/prelights.32219

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Author's response

Joshua I. Raji and Christopher J. Potter shared

  1. You mention that your study is a “snapshot” of the expression of ionotropic receptors in mosquitoes that are five-to-seven days old. Are there any plans to look at other age ranges? Could there be any interesting insights from looking at ionotropic receptor expression in larvae or pupae? And would your in situ probes be possible in larval antennae?

We captured IR expression in the antennae of adult female Anopheles at age 5-7 days post emergence (dpe) to coincide with the peak period of their host-seeking behavior. Yes, it would be interesting to compare this snapshot with early stage (1-3dpe) or late stage (9-11dpe) mosquitoes. In fact, we are extending our investigation to test if a blood meal alters IR spatial organization in the female antenna. Transcriptomics evidence from the Zwiebel lab suggests that a blood meal could modulate the abundance of Ionotropic receptors, and we’d like to see if the number of IR-expressing neurons or their organization may also be altered after ingesting a blood meal.

We are also curious to know which IRs are expressed at different developmental stages and which ones are turned on after emergence from the pupal stage. This could offer insights into which IRs are important at the aquatic stage of development. We have successfully optimized our in situ protocol to work on the mosquito pupal antennae. In situs have been performed on mosquito larval antennae and we think our current protocol should be adaptable.

  1. There are a lot of tantalizing instances of co-expression in your paper that seem like they could be worth follow-up studies, e.g., IR751 and IR8a being co-expressed suggests that IR751 is likely sensitive to acids. If you could only do functional studies on one of these combinations (empty neuron studies, for example), which combination would you investigate further, and why?

This is a great question! This was also one of our favorite aspects of the study—that we could use IR co-receptor co-expression to start predicting the general function of a tuning IR. We are very curious about IR75l and have now designed a CRISPR construct for this gene so we can make a knock-in. This will enable us to study the contribution of IR75l to odor detection in vivo. We are particularly interested in IR75l because it is likely to be an acid receptor, and we’d like to figure out which acids it might respond to. In general, we would like to examine the functional contributions of all the antennal IRs using an heterologous system and perform in vivo functional studies on the ones that appear to be the key players in host odor detection.

  1. Any speculation as to why ionotropic receptors are most expressed on the distal portion of the antenna and not the proximal portion?

We had the same question after observing the expression patterns. If we think of the antenna as the mosquito’s nose, it makes sense that many odor sensors would be concentrated towards the antennal tip which is the most exposed. This could facilitate detection or localization of volatile chemical compounds when they wave their antennae around. It would also be interesting to examine the function of IRs that appear to be proximally localized (like Ir41a and Ir75d )—that might give us clues as to why the antenna would not just have an even distribution of IRs along the antenna (as we had expected going into this work).

  1. Do you think that odorant receptors could share a similar pattern of distribution along the antenna?

Yes, especially if the distribution we observe for IRs is necessary for the function of the antenna as a whole. But it will require in situs of many antennal tuning ORs to be carried out to know for sure!

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