A persistent behavioral state enables sustained predation of humans by mosquitoes

  1. Trevor R Sorrells
  2. Anjali Pandey
  3. Adriana Rosas-Villegas
  4. Leslie B Vosshall

  • Curated by eLife

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    Evaluation Summary:

    This manuscript describes a female mosquito's behavior after a brief exposure to CO2, which has long been known to trigger host-seeking behaviour in female mosquitoes. The authors develop optogenetic tools in Aedes aegypti that enable the controlled delivery of 'fictive' CO2 to them. They show that a brief pulse of fictive CO2 alters the behavioral state of female mosquitoes, which lasts about 15 minutes. It provides new insights into how activation of CO2-sensing olfactory neurons alters the behavioral state of a mosquito towards sensory cues to increase host-seeking behaviors. The study will be of great value to the vector biology community, as well as to neurobiologists in general.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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Abstract

Predatory animals pursue prey in a noisy sensory landscape, deciding when to continue or abandon their chase. The mosquito Aedes aegypti is a micropredator that first detects humans at a distance through sensory cues such as carbon dioxide. As a mosquito nears its target, it senses more proximal cues such as body heat that guide it to a meal of blood. How long the search for blood continues after initial detection of a human is not known. Here, we show that a 5 s optogenetic pulse of fictive carbon dioxide induced a persistent behavioral state in female mosquitoes that lasted for more than 10 min. This state is highly specific to females searching for a blood meal and was not induced in recently blood-fed females or in males, who do not feed on blood. In males that lack the gene fruitless , which controls persistent social behaviors in other insects, fictive carbon dioxide induced a long-lasting behavior response resembling the predatory state of females. Finally, we show that the persistent state triggered by detection of fictive carbon dioxide enabled females to engorge on a blood meal mimic offered up to 14 min after the initial 5 s stimulus. Our results demonstrate that a persistent internal state allows female mosquitoes to integrate multiple human sensory cues over long timescales, an ability that is key to their success as an apex micropredator of humans.

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  1. Version published to 10.7554/elife.76663 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In the submitted manuscript, Sorrells and colleagues have characterized the shift in behavioral state female mosquitoes show after exposure to CO2. The authors have generated a mosquito line where CsChrimson is specifically expressed in the Gr3 expressing CO2 sensing neurons. Activation of these neurons through a 5s pulse of red light induced increased walking and probing behavior, which lasted 14 minutes.

    All in all, a very interesting and well-executed study. The topic is important, and the successful use of optogenetics in Aedes is nice to see! The text is easy to follow, the figures all acceptable. The schematic drawings of the setups are, however, not the prettiest...nor that easy to interpret.

    The submitted manuscript was accompanied by extensive reviewer comments from a previous submission. The main concern of those reviewers mostly centered around the novelty and broader importance of the work, issues which I believe are of no relevance here, or at least not to the same extent. The minor-ish technical concerns raised by the referees were all, in my view, addressed satisfactorily by the authors, and I see no reason for further experimentation. One point is perhaps worth repeating, namely whether the small size of the behavioral chambers could have influenced the results. Perhaps? It would clearly be interesting to see how the mosquitoes would behave in a larger arena. But that would be for another study.

    We agree that carrying out these experiments in a larger arena will be important, and also agree that it is out of scope for the current study.

    Reviewer #3 (Public Review):

    In this work, the authors aimed to use new genetic tools to control the activity of olfactory neurons that sense carbon dioxide. These genetic tools specifically express Chrimson (a red-light activated channel rhodopsin) only in CO2-sensing neurons in the maxillary palp of the mosquito. Using this method, the authors could use red light to activate the CO2-sensing neurons as if these neurons had been stimulated by CO2. This 'fictive' CO2 activation allowed the authors to carefully and temporally control when these neurons would be activated in relation to other sensory cues such as heat or the presence of a blood-meal. CO2-sensing neurons could also now be activated in the absence of air flow. This simplifies the sensory stimuli presented to the mosquito so that behaviors induced by CO2 sensory neuron stimulation can be examined without the complicating factor of persistent mechanosensory stimulations. The behavioral experiments and new assays are clever and well designed, and the authors present robust evidence that fictive CO2-sensory neuron stimulation leads to a persistent host-seeking state in the female mosquito. This activated state lasts for many minutes and influences such behaviors as probing and blood-feeding. The genetic tools and data analyses methods introduced here will allow the authors and others in the field to make advances into investigating how activation of CO2 sensory neurons leads to potent changes in the nervous system of the mosquito. This work further pioneers the use of optogenetics to link neurons and behaviors in a mosquito system and paves the wave for similar studies in other non-model insects.

    A weakness of the current work is the lack of direct neuronal activity measurements under the optogenetic stimulations. While the authors present strong evidence that their light stimulations can lead to behavior, it is not clear how these stimulations relate to activities induced by natural CO2 stimulations. These could be addressed by using their Gr3>Chrimson mosquitoes and performing single sensillum recordings from capitate peg sensilla (which house the CO2-sensing neurons), and examining how red light intensities change the activity of these neurons. This would ensure that the conditions used for fictive CO2 stimulations are a fair approximation of natural CO2 conditions.

    Thank you for this suggestion. We agree that direct recordings of neural activity would give a direct relationship between light and CO2 concentration. We do not feel our conclusions depend on knowing this, but it is an important area for future investigation.

    Alternatively, the authors could present evidence that their particular light stimulation parameters were chosen based on experimental behavioral experiments.

    Thank you for this suggestion. We have added to the manuscript a light dose-response curve in Figure 1—figure supplement 1. The proportion of mosquitoes responding increases with light intensity. Notably, the duration of the response is relatively independent of the light intensity (when a detectable number of mosquitoes respond). We added the following sentences to the results section:

    “The proportion of mosquitoes responding increased with light intensity (Figure 1—figure supplement 1B-D).”

    “Varying the light intensity changed the proportion of mosquitoes responding but not the duration of the response (Figure 1—figure supplement 1).”

    We also added additional details about the choice of intensity to the methods:

    “The light intensity chosen was an intermediate intensity as determined by a light-behavior dose-response curve (Figure 1—figure supplement 1B-D) .”

    “Red light stimuli were 627 nm at an intensity of 12 µW/mm2, chosen as an intermediate intensity that allowed the possibility of both an increase and decrease in the behavioral response.”

  3. eLife

    Evaluation Summary:

    This manuscript describes a female mosquito's behavior after a brief exposure to CO2, which has long been known to trigger host-seeking behaviour in female mosquitoes. The authors develop optogenetic tools in Aedes aegypti that enable the controlled delivery of 'fictive' CO2 to them. They show that a brief pulse of fictive CO2 alters the behavioral state of female mosquitoes, which lasts about 15 minutes. It provides new insights into how activation of CO2-sensing olfactory neurons alters the behavioral state of a mosquito towards sensory cues to increase host-seeking behaviors. The study will be of great value to the vector biology community, as well as to neurobiologists in general.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In the submitted manuscript, Sorrells and colleagues have characterized the shift in behavioral state female mosquitoes show after exposure to CO2. The authors have generated a mosquito line where CsChrimson is specifically expressed in the Gr3 expressing CO2 sensing neurons. Activation of these neurons through a 5s pulse of red light induced increased walking and probing behavior, which lasted 14 minutes.

    All in all, a very interesting and well-executed study. The topic is important, and the successful use of optogenetics in Aedes is nice to see! The text is easy to follow, the figures all acceptable. The schematic drawings of the setups are, however, not the prettiest...nor that easy to interpret.

    The submitted manuscript was accompanied by extensive reviewer comments from a previous submission. The main concern of those reviewers mostly centered around the novelty and broader importance of the work, issues which I believe are of no relevance here, or at least not to the same extent. The minor-ish technical concerns raised by the referees were all, in my view, addressed satisfactorily by the authors, and I see no reason for further experimentation. One point is perhaps worth repeating, namely whether the small size of the behavioral chambers could have influenced the results. Perhaps? It would clearly be interesting to see how the mosquitoes would behave in a larger arena. But that would be for another study.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript the authors develop an optogenetic tool to deliver 'fictive' CO2 to mosquitoes. This allowed them to unravel how CO2 can trigger host seeking behaviours in female mosquitoes. They validate their tool by demonstrating that it induces blood feeding in females that is comparable feeding induced by gaseous CO2. They then use it to make 3 important observations about the 'activating' quality of CO2:

    1. It induces a behavioural state that involves increased walking and probing and decreased flight. This state lasts for about 15 minutes and is specific to the activation of the CO2 sensory neurons: visual stimuli does not induce it and neither does activation of sweet-sensing neurons.
    2. This behavioural state is modulated just as blood-feeding behaviour is: blood-fed females and males don't show this behaviour, and, most interestingly, fru null males (who are attracted to vertebrate hosts) do!
    3. Host seeking involves multimodal sensory integration, and the authors use their tool to decipher aspects of this integration. They use heat as the second cue and address...
    1. ...whether the order of the two cues is important. They suggest that it is.
    2. ...how long the 'activation' ability of CO2 lasts. They suggest that heat needs to be presented about 4 minutes after CO2 for it to be integrated.
    3. ...for how long the integrated, persistent state lasts. They suggest that it lasts for 15 minutes.

    In summary, through this study, the authors have developed optogenetic tools in Aedes aegypti, which will be of use to a wider community, and have used it to advance our understanding of mosquito host-seeking behaviour. It will be of great value - both technologically and conceptually - to the community.

  6. eLife

    Reviewer #3 (Public Review):

    In this work, the authors aimed to use new genetic tools to control the activity of olfactory neurons that sense carbon dioxide. These genetic tools specifically express Chrimson (a red-light activated channel rhodopsin) only in CO2-sensing neurons in the maxillary palp of the mosquito. Using this method, the authors could use red light to activate the CO2-sensing neurons as if these neurons had been stimulated by CO2. This 'fictive' CO2 activation allowed the authors to carefully and temporally control when these neurons would be activated in relation to other sensory cues such as heat or the presence of a blood-meal. CO2-sensing neurons could also now be activated in the absence of air flow. This simplifies the sensory stimuli presented to the mosquito so that behaviors induced by CO2 sensory neuron stimulation can be examined without the complicating factor of persistent mechanosensory stimulations. The behavioral experiments and new assays are clever and well designed, and the authors present robust evidence that fictive CO2-sensory neuron stimulation leads to a persistent host-seeking state in the female mosquito. This activated state lasts for many minutes and influences such behaviors as probing and blood-feeding. The genetic tools and data analyses methods introduced here will allow the authors and others in the field to make advances into investigating how activation of CO2 sensory neurons leads to potent changes in the nervous system of the mosquito. This work further pioneers the use of optogenetics to link neurons and behaviors in a mosquito system and paves the wave for similar studies in other non-model insects.

    A weakness of the current work is the lack of direct neuronal activity measurements under the optogenetic stimulations. While the authors present strong evidence that their light stimulations can lead to behavior, it is not clear how these stimulations relate to activities induced by natural CO2 stimulations. These could be addressed by using their Gr3>Chrimson mosquitoes and performing single sensillum recordings from capitate peg sensilla (which house the CO2-sensing neurons), and examining how red light intensities change the activity of these neurons. This would ensure that the conditions used for fictive CO2 stimulations are a fair approximation of natural CO2 conditions. Alternatively, the authors could present evidence that their particular light stimulation parameters were chosen based on experimental behavioral experiments.

  7. preLights

    Excerpt

    old papers: mosquitoes follow you by tracking CO2 you exhale me: will hold my breath when a mosquito annoys me preprint: mosquitoes search for up to 14min after sensing CO2. me: 🤬

  8. Version published to 10.1101/2021.10.06.463436v2 on bioRxiv
  9. Version published to 10.1101/2021.10.06.463436v1 on bioRxiv