IRoning out mosquitoes’ attraction to mugginess

Mosquitoes spread the pathogens that cause an array of devastating diseases that afflict hundreds of millions of people each year. Among the most pernicious mosquito species is Aedes (Ae.) aegypti. The females hunt us down for our blood, since they need the nutrients for egg development. Unfortunately for us, we are their favorite host. If you are unlucky enough to be bitten after they had previously engorged on blood from someone infected with one of the viruses that cause dengue, yellow fever, chikungunya, or Zika, there is a high likelihood you will become infected. Another goal of the mosquito is to find an appealing source of standing water, like in a trash can or an abandoned car tire, where they can lay their fertilized eggs. Humidity sensing by mosquitoes is of particular interest since it functions in promoting effective host-seeking and oviposition. However, relative to other sensory cues that they detect, it has received only scant attention. In a new study, Tang et al. use state-of-the-art approaches to define two cation channels, called Ionotropic Receptors (IRs), that are each specifically required for function of just one of two antagonistic classes of cells for humidity detection (hygrosensation): Dry Cells or Moist Cells (1). The work provides key new insights into the cellular basis that enable Ae. aegypti to employ hygrosensation to home in on people from close range and to identify oviposition sites.

Anthropophilic female mosquitoes are exquisitely successful in finding us because they use multisensory integration. Hygrosensation is one of an array of human-derived stimuli that female mosquitoes employ to attack people with great precision. No single cue is sufficient. Of importance for long-range navigation is the mosquitoes’ keen ability to detect exhaled CO₂, even when we are many meters away. This increases their activity and motivates them to pay much greater attention to volatile human odors, as well as images of people, both of which can also be detected over multiple meters. Once the mosquitoes are <0.8 m away, they sense the thermal infrared emanating from our ~34 °C surface body temperature (2). When the females navigate to <10 centimeters from us, they start to pick up the convection heat from our bodies, as well as humidity generated by perspiration and breathing (3). After landing on our body, they sample nonvolatile cues. If they are attractive, they probe for a vein as they traverse our body surface.

It has been known for decades that anthropophilic mosquitoes sense humidity (4). However, until a seminal study published last year by the Garrity group (3), there were no reports describing the cellular and molecular mechanisms underlying mosquito hygrosensation. Nevertheless, work on many insects indicates that a small proportion of the antenna is devoted to responding to changes in humidity (5–7).

The importance of the insect antennae for sensing the environment is profound. In addition to assessing humidity, it functions in temperature sensation, olfaction, touch, hearing, and in monitoring wind and gravity. Each of these roles depends on neurons associated with sensilla. These are highly diverse sensory structures, some of which protrude from the antenna. Others lie nearly flat on the cuticle surface or internally at the bottom of a roundish depression (pit-in-pit sensilla) or at the end of thin tube (peg-in-tube sensilla, also called sensilla ampullacea) (4, 8). Insect hygrosensation depends on two types of neurons—one activated by dry air (Dry Cell) and another by moist air (Moist Cell) (5). These hygrosensory neurons allow insects to respond either to an increase in vapor pressure (Moist Cell) or a decrease (Dry Cell).

Until recently, we did not know the sensilla harboring the Moist Cells and Dry Cells in mosquitoes. In some insects, these cells are associated with external sensilla; however, internal sensilla have also been proposed to function in hygrosensation (5). In a major advance reported last year by the Garrity group, they established that Moist Cells and Dry Cells are embedded in peg-in-tube sensilla (sensilla ampullacea) near the base of the antenna in Ae. aegypti (Fig. 1 A–C) and in Anopheles gambiae, the vector for the malaria parasite (3). A goal of this study was to identify a receptor required for hygrosensation. So, they took advantage of prior discoveries in Drosophila demonstrating that several members of the large family of cation channels, IRs, including IR93a, are required for responding to dry and moist air (9–12). IRs are distant cousins of ionotropic glutamate receptors, and dozens are expressed in fruit flies, and mosquitoes, but not in mammals (13, 14).

Laursen et al. (3) interrupted the Anopheles and Aedes Ir93a, with a gene reporter, which was widely expressed in the antenna, including the first two of 13 flagellomeres, where it stained the sensilla ampullacea—candidate sensilla for containing the Dry Cells and Moist Cells. The investigators used transgenic mosquitoes expressing a calcium sensor (GCaMP) and found that two of the three cells in wild-type sensilla ampullacea responded either to moist or dry air. Mutation of Ir93a muted the responses of both classes of cells, impaired humidity seeking from a source at close range, diminished blood feeding, and resulted in reduced oviposition behavior.

The motivation for the latest work from the Garrity group (1) was to reveal the relative contributions of the Dry Cells and Moist Cells for hygrosensation. Drosophila homologs of Ir40a and Ir68a are specifically required for responses of the Dry Cells and Moist Cells (9–12). Therefore, Tang et al. used CRISPR to interrupt the Ae. aegypti Ir40a and Ir68a genes with reporters and found that each labeled just one of the three cells in the sensillum ampullacea in flagellomeres 1 and 2 (Fig. 1 A–C). By driving GCaMP in just one cell type, the authors demonstrated that Ir40a-positive cells respond to dry air (Dry Cells), while the Ir68a cells respond to moist air (Moist Cells).

The reporters also labeled two of the three cells in peg-in-pit sensilla on the sides of flagellomeres 10 and 12 (Fig. 1D; side coeloconic sensilla) and in tip coeloconic sensilla in flagellomere 13 (Fig. 1E). This was intriguing since coeloconic sensilla were primarily thought to function in thermosensation. Using GCaMP, Tang et al. established that the side coeloconic sensilla also harbor both Dry Cells and Moist Cells. However, the findings in tip coeloconic sensilla were different. While the Ir68a-positive cell was a Moist Cell, the Ir40a-expressing cell was not a Dry Cell but was responsive to warming. The thermal sensitivity of the Warming Cell was not dependent on Ir40a. Rather, the Warming Cell might be the neuron recently shown to respond to thermal infrared, and which depends on the TRPA1 channel, and two opsins for this response (2).

In addition to the three types of sensilla that function in hygrosensation (Fig. 1 C–E), might there be additional humidity-sensing sensilla? Several fruit fly olfactory sensilla sense humidity (15, 16), raising the possibility that some mosquito olfactory sensilla might also harbor hygrosensory cells. However, hygrosensory sensilla tend to be located in invaginations and are typically pore-less, while olfactory sensilla are external with many pores. The finding that a Drosophila mechanosensitive channel, TMEM63, is a hygrosensor (15) begs the question as to whether mosquito TMEM63 also functions in humidity detection.

A future issue is the mechanism through which Dry Cells and Moist Cells are activated. There are several models (17). According to one, changes in humidity alter the shape of sensilla, thereby activating a mechanosensitive channel. Second, humidity impacts the osmolarity of the sensilla lymph, which could also activate a receptor mechanically. Third, humidity is sensed by cooling due to evaporation. However, the cells defined by Tang et al. are unresponsive to temperature changes. To clarify whether hygrosensory IRs are mechanically activated, it would be informative to functionally express them in vitro and perform electrophysiology. This would require knowing the subunit composition of the receptors. IR93a might be a coreceptor that heteromultimerizes with IR40a in the Dry Cell and with IR68a in the Moist Cell. Another potential coreceptor is IR25a, which in Drosophila is broadly expressed and also functions in hygrosensation (9–12). Thus, both IR93 and IR25a might be coreceptors, while IR40a and IR68a serve as specificity subunits enabling the receptors to be tuned to dry and moist air, respectively. Since IRs are most likely tetramers (18), the receptors could each include an additional subunit, which remains to be identified.

Tang et al. also clarified the consequences on blood feeding and oviposition resulting from disrupting just the Dry Cell or Moist Cell. In contrast to the Ir93a mutant and to the Ir40a;Ir68a double mutant, which disrupt responses of the Dry Cell and Moist Cell, loss of just Ir40a or Ir68a did not diminish blood feeding. Thus, the requirement for Dry and Moist Cells for blood feeding is redundant. This indicates that either enhanced activity of the Moist Cell or suppressed responsiveness of the Dry Cell is sufficient to evaluate relative humidity in the context of blood feeding.

The Garrity group also analyzed the requirements for the Dry Cells and Moist Cells by offering females a choice between two containers—one that was dry and the other with water. As expected, wild-type females deposited their eggs exclusively in the water container, and as shown previously, the Ir93a mutant laid almost no eggs (3). However, the Ir40a mutant displayed normal oviposition, while the Ir68a females did not lay eggs. The results suggest that only Moist Cells are critical for oviposition but may not exclude the contribution of a yet unidentified Ir40a-independent Dry Cell.

Overall, the study by Tang et al. adds exciting new insights into the cellular and molecular mechanisms underlying hygrosensation in an anthropophilic mosquito. Of equal importance, the work also raises many intriguing new questions to IRon out in the future.