When they bite humans, mosquitoes can transmit dangerous diseases. For example, the Aedes aegypti mosquito spreads the viruses that cause yellow fever, Zika and dengue fever. Only the female mosquitoes feed on blood so they can obtain the nutrients they need to develop their eggs. Once they are ready, the insects lay their eggs in carefully selected sites where fresh water collects: if instead they choose places where the water is too salty, their offspring will die.

To find a suitable site, a mosquito ‘tastes’ the water by dipping in its legs and mouthparts, which activates the insect’s sensory neurons and sends signals to its brain. However, it remains unclear exactly how the mosquitoes can distinguish between fresh and salty water. To address this question, Matthews, Younger and Vosshall used a combination of genetic and imaging approaches to study female Ae. aegypti mosquitoes.

These experiments identified a gene known as ppk301 that is necessary for the mosquitoes to successfully lay their eggs in the right type of water. Mutant Ae. aegypti mosquitoes lacking the ppk301 gene did not properly lay eggs in fresh water even when given the opportunity.

Further experiments found that ppk301 was present in specific neurons in the legs and mouthparts of the mosquitoes. In leg neurons, ppk301 played a crucial role in sensing the presence of water and in stimulating the mosquitoes to lay eggs in water containing low levels of salt. However, these cells still responded to salt, even when lacking ppk301: other unidentified genes must therefore also be involved in preventing the mosquitoes from breeding in water that is too salty.

Every year, Aedes aegypti and other mosquitoes infect hundreds of millions of people and cause 500,000 deaths. Knowing exactly how mosquitoes breed could help to develop traps, repellents and other strategies to stop the insects from multiplying.

A female Ae. aegypti mosquito must take a blood-meal to develop a batch of eggs. Many strains of Ae. aegypti are anthropophilic, meaning that they target humans as their preferred blood source (Ponlawat and Harrington, 2005; McBride et al., 2014). Once proteins and other nutrients in the blood have been converted into eggs, a female mosquito must find a suitable body of freshwater to lay these eggs (Day, 2016). Larval and pupal stages of Ae. aegypti are aquatic and thus the choice of egg-laying site is a primary determinant of offspring survival (Christophers, 1960; Powell and Tabachnick, 2013). In the field, Ae. aegypti often lay eggs in small containers of freshwater, such as drinking water storage containers, discarded tires, clogged gutters, and other by-products of human settlement, likely contributing to their pernicious ability to adapt and colonize human settlements across the globe (Kraemer et al., 2015).

The act of choosing an egg-laying site in many insects, including Ae. aegypti, is a deliberate and evaluative behavior. A female Ae. aegypti mosquito with a fully developed batch of eggs will use elevated humidity and bacterial volatiles to locate water at a distance (Bentley and Day, 1989; Barbosa et al., 2010). Once in close-range, a mosquito contacts water to evaluate its suitability for egg-laying by sensing a wide variety of cues that include salinity, food, bitter toxins, and animal-derived chemical signals indicating the density of conspecific larvae and pupae or the presence of predators (Hudson, 1956; Benzon and Apperson, 1988; Bentley and Day, 1989; Zahiri and Rau, 1998; Pamplona et al., 2009; Afify and Galizia, 2015; Day, 2016; Zuharah et al., 2016). The physiological mechanisms by which a mosquito senses the presence of water and evaluates its composition to safely nurture her offspring remain unknown.

Here, we show that the DEG/ENaC channel ppk301 is required for mosquitoes to exploit freshwater egg-laying substrates. When ppk301 mutant females contact water, they do not lay eggs as readily as wild-type animals and are more likely to make aberrant decisions between freshwater and saltwater at concentrations that impair offspring survival. We developed a CRISPR-Cas9-based genetic knock-in strategy to build genetic tools for labeling and imaging molecularly defined populations of neurons and generated a reporter line expressing the QF2 transcriptional activator from the endogenous ppk301 locus. We found that ppk301 is expressed in sensory neurons in legs and proboscis, appendages that directly contact water, and that sensory afferents from these ppk301-expressing neurons project to central taste centers. Using in vivo calcium imaging with the genetically encoded calcium sensor GCaMP6s at the axonal terminals of ppk301-expressing neurons in the ventral nerve cord, we found that ppk301 neurons respond to freshwater and that this response was almost entirely abolished in ppk301 mutant animals. Surprisingly, these projections were also activated by salt in both wild-type and ppk301 animals. We propose that ppk301 drives egg-laying at low-salt concentrations and that egg-laying is inhibited by a ppk301-independent pathway at high-salt concentrations.

We observed Ae. aegypti egg-laying behavior in the laboratory to understand the range of sensory information that is integrated to guide the final decision to lay an egg. A female Ae. aegypti mosquito with a fully developed batch of eggs will first evaluate a potential egg-laying site by physical contact with some combination of sensory tissues that include all three pairs of legs and her proboscis. If a site is deemed suitable, she will lay eggs singly on the moist substrate above the waterline (Hudson, 1956) (Figure 1A, Video 1). When we used a mesh barrier to block access to liquid water, females did not lay eggs, even though they had free access to the moist filter paper substrate (Figure 1B,C). Together, this suggests that direct contact with liquid water is necessary to stimulate egg-laying in Ae. aegypti and we set out to probe the molecular and cellular basis of this water-sensation to better understand this critical mosquito behavior.

Figure 1

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Video 1

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As a consequence of their global spread, Ae. aegypti are faced with diverse habitats with a wide variety of potential egg-laying sites. For example, they can be found in abundance in a number of coastal regions rich with standing saltwater (Ramasamy et al., 2014; de Brito Arduino et al., 2015). To mimic the choice between freshwater and seawater in the lab, we developed a two-choice assay in which individual blood-fed females were placed in a container with a divided Petri dish filled with deionized water on one side and varying concentrations of a chemically defined artificial seawater solution (Kester et al., 1967) on the other (Figure 1D, Supplementary file 1). Mosquitoes showed no significant preference between deionized water and dilute seawater up to 10%, with individual mosquitoes either picking a solution at random or distributing their eggs between both solutions (Figure 1D). However, they showed a strong dose-dependent aversion to higher concentrations of seawater, with an IC₅₀ of 12.25% seawater (Figure 1D,E). Females showed near-complete aversion to 25–100% seawater (Figure 1D,E). These choices have consequences for the offspring. When we measured survival of offspring reared in varying concentrations of seawater, we found dose-dependent lethality (LD_50 = 25.23%) (Figure 1E). To simplify the stimulus, we used sodium chloride (NaCl), the predominant salt in artificial seawater, in all subsequent experiments. Females showed dose-dependent inhibition of egg-laying with increasing concentrations of NaCl (Figure 1F) when they were only given access to a single concentration, suggesting that the preference for freshwater may be driven in part by an aversion to laying eggs in saltwater. Similar to artificial seawater, NaCl produced a dose-dependent decrease in offspring survival (Figure 1G). This demonstrates that the female mosquito’s choice of freshwater or saltwater correlates with the survival of her offspring, making this an essential decision for the propagation and fitness of the species.

In a search for genes that control Ae. aegypti freshwater egg-laying, we reasoned that animals carrying a mutation in an egg-laying preference gene would fail to lay eggs in freshwater, inappropriately lay in saltwater, or both. pickpocket (ppk) genes are members of the Degenerin/ENaC channel superfamily and encode cation channel subunits that function as putative mechanoreceptors and chemoreceptors for a wide array of stimuli including pheromones, liquid osmolality, and salt (Adams et al., 1998; Chalfie, 2009; Cameron et al., 2010; Chen et al., 2010; Zelle et al., 2013). Through manual curation of geneset annotations, we predict that the Ae. aegypti genome (Matthews et al., 2018) contains 31 ppk genes. These are unevenly distributed across its three chromosomes. Whereas chromosome 1 has only three ppk genes, chromosome 3 has 22, including three clusters of 3–7 ppk genes (Figure 2A). The predicted protein products of many ppk genes have clear 1-to-1 orthologues in Drosophila melanogaster and the malaria mosquito Anopheles gambiae, while others are in species-specific expansions (Figure 2B). Examination of tissue-specific transcript abundance using previously published RNA-seq data (Matthews et al., 2016; Matthews et al., 2018) reveals a broad range of expression patterns in adult tissues, including in legs, proboscis, and other sensory tissues (Figure 2C). In a previous study (Kistler et al., 2015), we generated CRISPR-Cas9 mutations in four ppk genes (marked with arrowheads in Figure 2A,B) that we considered as candidates for controlling egg-laying.

Figure 2

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We first measured the ability of these mutant strains to blood-feed and found that all four were attracted to and engorged fully on the blood of a live human host (Figure 3A). To test egg-laying behavior, we introduced single blood-fed females of each strain into egg-laying vials containing a small amount of water and a filter paper as an egg-laying substrate. One mutant, ppk301, an orthologue of the Drosophila melanogaster ppk28 low-osmolality sensor (Cameron et al., 2010; Chen et al., 2010), showed a defect in egg-laying. Fewer than 40% of ppk301 mutants laid more than 10 eggs (Figure 3B). To exclude the possibility that this defect was due to an inability to convert blood into developed embryos, we counted the number of mature eggs in ovaries and confirmed that there was no difference between wild-type and ppk301 mutants (Figure 3C).

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To investigate freshwater egg-laying preference in the ppk mutants, we introduced single blood-fed female mosquitoes into individual chambers containing two Petri dishes, one filled with freshwater (0 mOsm/kg NaCl) and the other with 200 mOsm/kg NaCl. Even when ppk301 mutant animals laid eggs, they laid fewer eggs than wild-type on water and more eggs than wild-type on 200 mOsm/kg NaCl (Figure 3D). We assayed these four ppk mutant strains across a range of NaCl concentrations and found a significant reduction in aversion to salt solution only in the ppk301 mutant, as measured by the proportion of animals laying eggs primarily on salt solution (Figure 3D–F). Together, these data support the conclusion that mutations in ppk301 disrupt freshwater egg-laying in two distinct ways: by dramatically reducing the drive to lay eggs in suitable, low-salt, substrates and reducing the aversion to concentrations of NaCl that are lethal to their offspring.

If ppk301 mutant animals fail to sense water, which normally triggers mosquitoes to lay an entire clutch of eggs in a short timespan, we hypothesized that these mutants would show a delay in onset and a reduced egg-laying rate when housed in close proximity to water for many days. To ask if ppk301 mutant animals will lay eggs on water given sufficient time, we introduced individual blood-fed females into egg-laying vials containing water and monitored the number of eggs laid per female per day over 7 days. While the vast majority of wild-type and ppk301 heterozygous animals laid all their eggs within the first 2 days of being introduced into egg-laying vials, ppk301 mutant animals did not. Instead, the mutants showed increased variability in the time of egg-laying initiation and a tendency to spread egg-laying out over many days (Figure 3G–J). This slow, sustained, and variable egg-laying behavior on freshwater is consistent with a defect in sensing the water that triggers egg-laying.

If ppk301 directly senses the osmolality or salinity of liquid, we would expect it to be expressed in the sensory appendages that contact water. At the inception of this project, genetic tools for labeling, monitoring, and manipulating neurons in Ae. aegypti did not exist. To address this gap, we developed new genetic tools in the mosquito to label all ppk301-expressing neurons and to image neuronal activity at sensory neuron terminals using the genetically encoded calcium sensor GCaMP6s (Chen et al., 2013). We adapted an approach in which a T2A ‘ribosomal skipping’ peptide is used to express multiple independent protein products from a single RNA transcript (Diao and White, 2012; Daniels et al., 2014). We first tested the efficiency of T2A in Ae. aegypti by generating a transgene containing a membrane-targeted mCD8:GFP fusion protein and a nuclear-targeted dsRed:NLS fusion protein separated by T2A and driven from the Ae. aegypti polyubiquitin promoter (Anderson et al., 2010) (Figure 4A). Confocal microscopy revealed complete subcellular separation of the two fluorophores in individual larval body-wall cells (Figure 4A). This demonstrates that T2A functions efficiently to prevent peptide bond formation in Ae. aegypti and can be used as a tool to independently express multiple gene products from a single locus.

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To build a flexible system for expressing a diverse array of effector transgenes, we employed the Q-binary expression system for transgene amplification (Potter et al., 2010), which has been successfully implemented in An. gambiae malaria mosquitoes (Riabinina et al., 2016; Afify et al., 2019). We used CRISPR-Cas9 with the same guide RNA used to generate the ppk301 mutant to introduce an in-frame T2A sequence into the ppk301 locus followed by the QF2 transcriptional activator (Figure 4B). This is predicted to cause a loss-of-function mutation in ppk301 and also express QF2 in all ppk301-expressing cells.

We also generated two transgenic QUAS effector strains. The first is a QUAS response element driving the expression of both cytosolic dTomato and GCaMP6s (15x-QUAS-dTomato-T2A-GCaMP6s), which allows us to simultaneously label neurons and image their activity with the genetically encoded calcium sensor GCaMP6s (Chen et al., 2013) (Figure 4C). The second drives expression of membrane-bound GFP (15x-QUAS-mCD8:GFP), allowing us to reveal the complete morphology of the neurons in which it is expressed (Figure 4I). We looked for expression of the ppk301>dTomato-T2A-GCaMP6s reporter in the appendages that contact water during egg-laying (Figure 4D) and found that sensory neurons innervating trichoid sensilla in the labellar lobes of the proboscis (Figure 4E) were labeled. We also found labeling in the legs, primarily in the distal tarsal segments (Figure 4F).

The mosquito central nervous system consists of a brain and a ventral nerve cord (Ito et al., 2014; Smarandache-Wellmann, 2016; Court et al., 2017) (Figure 4G). To facilitate our understanding of the neuroanatomy of Ae. aegypti in general, and the projection pattern of the ppk301 driver line in particular, we built a three-dimensional female mosquito brain atlas, in which we identified and named the major neuropils in accordance with nomenclature established by the Insect Brain Name Working Group (Ito et al., 2014) (Figure 4H and http://mosquitobrains.org). Projections of ppk301-expressing sensory neurons in the head extend processes specifically to the subesophageal zone (Figure 4H,J). Two nerves enter each hemisphere of the subesophageal zone, arising from the proboscis and the pharynx (Figure 4J). Additionally, each leg sends projections into the ventral nerve cord, with nerves running into each neuromere (Figure 4K). Both brain and ventral nerve cord innervation patterns are consistent with these neurons mediating taste sensation (Scott, 2018). We noted that projections of ppk301-expressing neurons are also present in the male brain and ventral nerve cord (Figure 4—figure supplement 1A–C), consistent with a role for ppk301 in behaviors other than egg-laying. Labeling in both males and females was absent in genetic controls expressing either QF2 driver or QUAS effector alone (Figure 4—figure supplement 1D–K).

We hypothesized that if ppk301-expressing neurons were promoting egg-laying, they should be maximally activated by water, and we set out to test this by functional calcium imaging in response to freshwater and behaviorally relevant concentrations of NaCl. We developed an in vivo calcium imaging preparation with GCaMP6s (Chen et al., 2013) in ventral nerve cord sensory afferents of a mosquito presented with water or NaCl solutions on a single foreleg (Figure 5A,B). This appendage was chosen because it most frequently contacts water during egg-laying (Figure 1A, Video 1). We imaged the prothoracic segment of the ventral nerve cord with two-photon microscopy, using a custom fluidics device to deliver and retract liquids to the foreleg and compared responses to different stimuli within individual animals. ppk301-expressing neuronal projections in the ventral nerve cord were identified by dTomato expression (Figure 5C), which was also used to determine the region of interest for calcium imaging analysis.

Figure 5

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We observed low GCaMP6s fluorescence at baseline and an increase in fluorescence in every trial where water was presented (Figure 5C–E), with no apparent desensitization across trials (data not shown). Only the ipsilateral side of the ventral nerve cord innervated by the stimulated leg showed activation (data not shown). In D. melanogaster, cells that express the ppk301 orthologue ppk28 respond to water but are inhibited by high osmolality solutions including NaCl (Cameron et al., 2010; Chen et al., 2010; Jaeger et al., 2018). The population response in ppk301-expressing afferents in the mosquito is functionally different, showing robust responses to water and strong activation by salt solutions (Figure 5D–F). To determine whether ppk301 is required for the neuronal responses to water or salt, we performed imaging experiments in a ppk301 mutant background and found that the response to water is almost entirely abolished in the ppk301 mutant (Figure 5D–F). In contrast, 200 and 300 mOsm/kg NaCl solutions still elicited a strong response in the ppk301 mutant, albeit with a small reduction in amplitude (Figure 5D–F). These data show that ppk301 is required for the activation of these neurons by freshwater and that a ppk301-independent pathway activates these neurons in response to salt. This suggests that mosquito ppk301-expressing afferents are either multi-modal (Zocchi et al., 2017) and each neuron responds to both water and salt, or that there is a functionally heterogeneous population of ppk301-expressing afferents with distinct neurons responding to water and salt.

To test if ppk301-expressing neurons are activated by any solution with increased osmolality, we performed imaging experiments with two additional solutes that are not ionic salts, L-serine and D-(+)-cellobiose. We delivered all stimuli at 200 mOsm/kg, the osmolality of NaCl that elicited the peak response. We found that ppk301-expressing neurons did not respond more strongly to L-serine or D-(+)-cellobiose than they did to water alone (Figure 6A–B). Thus, activity in these neurons does not only track with osmotic pressure. Intrigued by these findings, we performed egg-laying preference assays where mosquitoes were given a choice to lay eggs on freshwater and either L-serine or D-(+)-cellobiose. Although female mosquitoes found 200 mOsm/kg NaCl highly aversive, they did not discriminate between freshwater and L-serine or D-(+)-cellobiose at 200 mOsm/kg (Figure 6C–D). Thus, the behavioral aversion to salt is unrelated to osmotic pressure.

Figure 6

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The observation that ppk301-expressing cells are activated by both water and salt is intriguing because wild-type females avoid laying eggs specifically in high-salt solutions. To understand how female mosquitoes interact with these different stimuli, we monitored real-time behavior of individual females offered either water or 300 mOsm/kg NaCl over 40 min by scoring their contact with liquid and individual egg-laying events. Both wild-type and ppk301 mutant mosquitoes contacted water, but only wild-type females consistently transformed these touches into egg-laying events (Figure 7A). When offered 300 mOsm/kg salt solution, both wild-type and ppk301 mutant mosquitoes touched liquid, but neither genotype reliably laid eggs (Figure 7A). These results are consistent with a model (Figure 7B) in which activation of ppk301 cells by water is a permissive signal for rapid and reliable egg-laying. Animals lacking ppk301 fail to detect the water activation signal and show delayed and intermittent egg-laying. Activation of ppk301-expressing neurons by high salt is not sufficient to drive egg-laying, suggesting that ppk301-expressing cells gate egg-laying at low NaCl concentrations, but as NaCl concentrations increase, an independent noxious salt-sensing pathway is recruited that overrides the activation of the tarsal ppk301-expressing cells imaged in this study to inhibit egg-laying (Figure 7B). We predict mutations that disrupt this noxious salt sensor would yield mosquitoes that show indiscriminate egg-laying on a high-salt substrate.

Figure 7

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Ae. aegypti mosquitoes preferentially lay eggs in freshwater and avoid saltwater. We annotated the ppk ion channel gene family in Ae. aegypti and made targeted mutations in several candidate genes, identifying a mutation in ppk301 that disrupts freshwater egg-laying preference and behavior. ppk301 is expressed in sensory neurons of the tarsi and proboscis, tissues that contact water during egg-laying. ppk301-expressing tarsal sensory neurons are activated by both water and high salt. This response is not driven solely by osmotic pressure, because two other solutes L-serine and D-(+)-cellobiose presented at the same osmolality were indiscriminable from water. In a ppk301 mutant background, responses to water are almost entirely abolished but responses to salt remain. Together, these data suggest a model in which activation of ppk301-expressing neurons by water is required for egg-laying while a ppk301-independent pathway encodes aversion to high concentrations of salt (Figure 7B). This study identifies an important component of the circuitry regulating egg-laying behavior and provides an entry point into understanding the most important parenting decision a female mosquito makes.

What sensory cues and modalities activate ppk301-expressing neurons? Sensory neurons in the D. melanogaster proboscis that express the orthologous ppk28 gene are maximally activated by water but inhibited by dissolved solutes (Cameron et al., 2010; Chen et al., 2010; Jaeger et al., 2018). In contrast, the afferents of the ppk301-expressing cells in the mosquito tarsi are not tuned specifically to osmolality but rather encode multimodal responses to liquid (ppk301-dependent) and to high concentrations of salt (ppk301-independent). Whether or not individual neurons within this population exhibit distinct tuning properties remains to be determined. In addition to the tarsi, ppk301 is expressed in the proboscis and pharynx. It will be important to evaluate whether these populations of ppk301-expressing cells are strictly tuned to water, as are D. melanogaster ppk28-expressing labellar sensory neurons, and lack the multimodal responses seen in the tarsal ppk301-expressing neurons.

How does ppk301 regulate egg-laying? We favor a model in which ppk301-dependent activation of sensory neurons by liquid provides a permissive signal required for egg-laying, while high concentrations of salt activate noxious salt pathway(s) that can override this signal and prevent egg-laying. Together, these opposing signals are integrated to sculpt egg-laying preference and allow a mosquito to exploit freshwater substrates while preventing eggs from being laid on high-salt substrates that are lethal to their offspring.

It will be interesting to discover the biophysical mechanisms by which ppk301 neurons respond to water. ppk301 encodes a single subunit of a trimeric DEG/ENaC channel (Jasti et al., 2007). One possibility is that ppk301 ion channels are homomers that respond to osmotic pressure, similar to D. melanogaster ppk28 (Cameron et al., 2010; Chen et al., 2010). Alternatively, ppk301 could encode a promiscuous subunit that could be part of multiple heteromeric PPK channels with different ligands that account for some of the multimodal tuning properties seen in ppk301-expressing tarsal sensory neurons. In this scenario, genetic ablation of ppk301 could result in a novel set of PPK channels that are composed of the remaining subunits, thereby shifting the composition of channels on the membrane and influencing overall neuronal tuning properties.

Water- and salt-sensing are critically important because all animals must regulate fluid and ion homeostasis for the proper functioning of their physiological systems. Terrestrial insects such as mosquitoes are small and have a large surface-area-to-volume ratio, meaning that they are susceptible to desiccation. Therefore, both male and female adult mosquitoes must seek out liquid for survival. It is not known if sensory systems used for determining the physical properties of liquids during fluid ingestion in male and female mosquitoes are the same as those used by the female for evaluating water quality during egg-laying. Our data show that ppk301 is expressed in both male and female appendages, and that neurons expressing ppk301 project to similar areas of the brain and ventral nerve cord. Studying the effects of ppk301 mutations on liquid ingestion in adult Ae. aegypti will provide insight into whether the same molecules and sensory neurons are being used in distinct behavioral contexts. It will also be interesting to investigate if the activity of ppk301 neurons is modulated by physiological state, including the presence of developed eggs or elevated thirst due to dehydration or desiccation stress. Previous studies have shown the existence of a canonical ‘water’ cell in the apical labral chemoreceptor sensilla of Ae. aegypti (Werner-Reiss et al., 1999), but we do not see ppk301 expression in this tissue, indicating that there may be additional osmosensitive receptors encoded in the mosquito genome.

A complete cellular and molecular understanding of salt sensation in mosquitoes remains to be elucidated and the ‘noxious salt sensor’ proposed in our model remains to be discovered. We note that a distributed coding of salt taste in which distinct populations of sensory neurons are modulated by salt is not unprecedented. A recent study in Drosophila reveals that in the proboscis, salt can activate or inhibit every class of gustatory receptor neuron (Jaeger et al., 2018). Salt is also a key component of blood and understanding the mechanisms of salt sensation during blood-feeding, egg-laying, and other critical mosquito behaviors is an important future goal (Werner-Reiss et al., 1999; Sanford et al., 2013).

The evolution of egg-laying preference both within Ae. aegypti and in other mosquito species is a critical determinant of the types of environments and ecological niches that they are able to exploit. Indeed, some strains of Ae. aegypti have begun to exploit brackish water for egg-laying (Ramasamy et al., 2014; de Brito Arduino et al., 2015). Such an adaptation requires co-evolution of the shifting preference of females for salty water and larval tolerance of high salinity environments into which they are deposited. Investigating the underlying genetic changes that support the coordination of these shifting phenotypes in both larvae and adults will be important for understanding how Ae. aegypti adapts to novel breeding sites across the globe, which has greatly increased their capacity as a disease vector.

The advances described here depended critically on new genetic tools we developed that did not previously exist in this non-model insect. These techniques now make it possible to visualize neuronal anatomy and activity within molecularly identified cell types and to identify the genetic and neural circuit substrates of many mosquito behaviors. These behaviors contribute to the spread of deadly pathogens and understanding their underlying biology could contribute to mosquito control efforts. The development of similar tools and reagents in other insect species of public health, agricultural, or ethological interest will broaden our view of insect biology and facilitate comparative studies of the genes and circuits underlying evolutionary adaptations in insects.

Source data for all plots in Figures 1-3 and Figure 5-7 are provided in Supplementary file 1.

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Author details

1. Benjamin J Matthews

   1. Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, United States
   2. Howard Hughes Medical Institute, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Meg A Younger

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8697-699X

2. Meg A Younger

   1. Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, United States
   2. Kavli Neural Systems Institute, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Benjamin J Matthews

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4967-7939

3. Leslie B Vosshall

   1. Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, United States
   2. Howard Hughes Medical Institute, New York, United States
   3. Kavli Neural Systems Institute, New York, United States

   Contribution

   Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   leslie@rockefeller.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6060-8099

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