Author response: The cation channel TRPA1 tunes mosquito thermotaxis to host temperatures (2015)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract While most animals thermotax only to regulate their temperature, female mosquitoes are attracted to human body heat during pursuit of a blood meal. Here we elucidate the basic rules of Aedes aegypti thermotaxis and test the function of candidate thermoreceptors in this important behavior. We show that host-seeking mosquitoes are maximally attracted to thermal stimuli approximating host body temperatures, seeking relative warmth while avoiding both relative cool and stimuli exceeding host body temperature. We found that the cation channel TRPA1, in addition to playing a conserved role in thermoregulation and chemosensation, is required for this specialized host-selective thermotaxis in mosquitoes. During host-seeking, AaegTRPA1-/- mutants failed to avoid stimuli exceeding host temperature, and were unable to discriminate between host-temperature and high-temperature stimuli. TRPA1-dependent tuning of thermotaxis is likely critical for mosquitoes host-seeking in a complex thermal environment in which humans are warmer than ambient air, but cooler than surrounding sun-warmed surfaces. https://doi.org/10.7554/eLife.11750.001 eLife digest Temperature can vary considerably in an environment. Living organisms have evolved sensory systems to detect and avoid excessive heat or cold: a behavior that is termed 'thermotaxis'. In rare cases, animals use this ability to locate food sources in their environment. One example of such an adaptation is the female mosquito of the species Aedes aegypti. When a mosquito needs blood to produce her eggs, she becomes attracted to the body heat of warm-blooded hosts. But the range of temperatures that these mosquitoes prefer and the genes required for this behavior had not been been defined. Now, Corfas and Vosshall have found that female Aedes aegypti are highly sensitive to differences in temperature, and are capable of heat-seeking in a range of environmental temperatures. Furthermore, by seeking out things that are warmer than their surroundings, while avoiding those that are cooler or much hotter than their host's body temperatures, these mosquitoes tune their thermotaxis toward targets that resemble a human to feed upon. Corfas and Vosshall also discovered that a protein called TRPA1 is required for this tuning of Aedes aegypti's heat-seeking behavior. This protein is known to allow insects to detect chemical signals and regulate their own temperature, but it was not previously known that this protein was involved in mosquito thermotaxis. Mutant mosquitoes without the gene for TRPA1 failed to avoid high temperatures, which meant that they could no longer tell the difference between an overly hot target and a warm one that resembled their hosts. Following on from this work, the next challenge will be to characterize all the genes, sensory organs, and neural circuits that drive mosquito heat-seeking behavior. These findings may in the future inform the design of the next generation of repellents and traps for the control of mosquito-borne diseases, such as dengue and yellow fever. https://doi.org/10.7554/eLife.11750.002 Introduction Thermotaxis is a sensory-motor behavior that guides animals toward a preferred temperature. This type of sensory navigation allows animals to avoid environments of noxious cold and heat, with the goal of remaining in physiologically suitable ambient temperatures. For ectotherms, such as most insects, thermotaxis behavior is the primary method of thermoregulation. Terrestrial invertebrates are vulnerable to temperature extremes, facing the risk of desiccation at elevated temperatures, and rapid hypothermia at low temperatures. Therefore, mechanisms to detect environmental temperatures and trigger appropriate approach or avoidance behaviors are extremely important for their survival. For instance, adult Caenorhabditis elegans worms migrate preferentially toward a specific thermal environment determined by the temperature of their cultivation (Hedgecock and Russell, 1975; Mori and Ohshima, 1995). Adult Drosophila melanogaster flies prefer a narrow range of air temperatures around 24–25°C (Sayeed and Benzer, 1996; Hamada et al., 2008) and rapidly avoid air temperatures of ~31°C (Ni et al., 2013). Interestingly, some hematophagous (blood-feeding) arthropods have evolved a specialized mode of thermotaxis to locate endothermic (warm-blooded) hosts. Such thermophilic behavior is seen in kissing bugs [Triatoma infestans (Flores and Lazzari, 1996) and Rhodnius prolixus (Schmitz et al., 2000)], the bedbug [Cimex lectularius (Rivnai, 1931)], the tick [Ixodes ricinus (Lees, 1948)], and many species of mosquito (Clements, 1999) including Ae. aegypti, a major tropical disease-vector (Bhatt et al., 2013). Female Ae. aegypti require a vertebrate blood meal for the production of eggs, and finding a suitable warm-blooded host is therefore an essential component of reproduction. Mosquitoes use a variety of physical and chemical senses to locate hosts in their environment (Cardé, 2015). When host-seeking, these animals become strongly attracted to inanimate warm objects, eagerly probing at them as if they were hosts (Howlett, 1910). In nature, mosquitoes thermotax in a complex thermal landscape in which ambient air temperature, host body temperature, and surrounding surface temperatures can vary widely. For mosquitoes such as Ae. aegypti, host-seeking behavior can be activated by an increase in ambient carbon dioxide (CO2) (Majeed et al., 2014). This activation elicits flight activity (Eiras and Jepson, 1991; McMeniman et al., 2014) and results in an array of behaviors including attraction to visual stimuli (van Breugel et al., 2015) and host olfactory cues (Dekker et al., 2005; McMeniman et al., 2014), and landing on warm objects (Burgess, 1959; Eiras and Jepson, 1994; Kröber et al., 2010; Maekawa et al., 2011; McMeniman et al., 2014; van Breugel et al., 2015). Ae. aegypti flying in a wind tunnel can detect a warmed stimulus from a distance, eliciting attraction and thermotaxis (van Breugel et al., 2015). What are the mechanisms by which animals detect thermal stimuli, and how might these be adapted for the specialized needs of heat-seeking female mosquitoes? Thermotaxis is typically initiated by thermosensitive neurons that sample environmental temperature to inform navigational decision-making. Such neurons must be equipped with molecular thermosensors capable of detecting and transducing thermal stimuli. Diverse molecular thermoreceptors have been identified in the animal kingdom, many of which are members of the transient receptor potential (TRP) superfamily of ion channels (Barbagallo and Garrity, 2015; Palkar et al., 2015). Different thermosensitive TRPs show distinct tuning spanning the thermal spectrum from noxious cold to noxious heat. Among these is TRPA1, which is a heat sensor in multiple insects, including the vinegar fly D. melanogaster and the malaria mosquito Anopheles gambiae (Hamada et al., 2008; Wang et al., 2009). Neurons in thermosensitive sensilla (Gingl et al., 2005) of An. gambiae female antennae express TRPA1 (Wang et al., 2009). In D. melanogaster, TRPA1 is expressed in internal thermosensors located in the brain, and DmelTRPA1-/- mutants fail to avoid high air temperature in a thermal gradient (Hamada et al., 2008). Interestingly, some snakes and vampire bats express thermosensitive TRP channels in organs used to sense infrared radiation from warm-blooded prey (Gracheva et al., 2010; 2011). This raises the possibility that AaegTRPA1 may be used by mosquitoes to find hosts. Recently, a structurally distinct insect thermosensor, Gr28b, was identified in D. melanogaster (Ni et al., 2013). Gr28b, a gustatory receptor paralog, is expressed in heat-sensitive neurons of D. melanogaster aristae and is an important component of thermotaxis during rapid avoidance of heat (Ni et al., 2013). It is also highly conserved among Drosophila species (McBride et al., 2007), and has a clear ortholog in Ae. aegypti, AaegGr19 (Ni et al., 2013). A functional role for these thermosensors has never been investigated in the mosquito. Here, we use high-resolution quantitative assays to examine the behavioral strategies underlying mosquito heat-seeking behavior. Our results show that by seeking relative warmth and avoiding both relative cool and high temperatures, female mosquitoes selectively localized to thermal stimuli that approximate warm-blooded hosts. Using genome editing, we generated mutations in the candidate thermoreceptors, AaegTRPA1 and AaegGr19. We found that TRPA1 is required for tuning mosquito thermotaxis during host-seeking. AaegTRPA1-/- mutants lacked normal avoidance of thermal stimuli exceeding host body temperatures, resulting in a loss of preference for biologically relevant thermal stimuli that resemble hosts. This work is important because it identifies a key mechanism by which mosquitoes tune their thermosensory systems toward human body temperatures. Results We previously described an assay to model heat-seeking behavior in the laboratory by monitoring mosquitoes landing on a warmed Peltier element in the context of a cage supplemented with CO2 (Figure 1A,B) (McMeniman et al., 2014). This assay has the advantages that it is simple in design, produces robust behaviors, and enables the collection of data from large numbers of animals in a short experimental timeframe. Using this system, we can examine mosquito responses to diverse thermal stimuli and measure thermotaxis in different ambient temperature environments. We first needed to determine whether heat-seeking behavior habituates over multiple thermal stimulations. In our heat-seeking assay, Ae. aegypti mosquitoes reliably responded to 12 serial presentations of a 3-minute long 40°C stimulus over the course of more than 2.5 hr, with no evidence of habituation (Figure 1—figure supplement 1). Figure 1 with 1 supplement see all Download asset Open asset Mosquitoes thermotax to stimuli approximating host body temperature. (A) Schematic of heat-seeking assay enclosure (30 × 30 × 30 cm). (B) Representative experimental image showing mosquitoes detected on and near the Peltier (red square). (C) Typical skin and core temperatures of Ae. aegypti hosts, humans and chickens (Richards, 1971; Yao, et al., 2008. (D–F) Heat-seeking behavior measured for a range of stimuli from 26 to 60°C (n = 6 trials per condition). Peltier temperature measured by thermocouple (D, top trace, mean in red, s.e.m. in gray) and percent of mosquitoes on Peltier (D, bottom trace, mean in black, s.e.m. in gray). We note that variance in both traces is low, making s.e.m. traces difficult to see. (E) Percent mosquitoes on Peltier during seconds 90–180 of each stimulus period in (D). Each replicate is indicated by a dot, and mean by a line. Arrowheads indicate significant differences (p < 0.05) from the second presentation of the 40°C stimulus or from 26°C (repeated measures one-way ANOVA with Bonferroni correction). (F) Heat maps showing mean mosquito occupancy on the Peltier (red square) and surrounding area, during seconds 90–180 of each stimulus period in (D). Bold borders indicate stimuli with responses significantly different from 26°C stimulus (top row) or 40°C stimulus (bottom row) in (E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). https://doi.org/10.7554/eLife.11750.003 Ae. aegypti can feed on a variety of hosts (Clements, 1999; Tandon and Ray, 2000) with core body temperatures ranging from ~37°C (humans) to ~40–43°C (chickens) (Richards, 1971) (Figure 1C). It is unknown whether there are minimal or maximal temperature thresholds constraining mosquito heat-seeking, and whether responses to thermal stimuli depend on the background ambient temperature. To investigate these questions, we measured attraction to thermal stimuli produced by heating the Peltier to temperatures ranging from ambient (set to 26°C in these experiments) to 60°C (Figure 1D, E). We found that mosquitoes were highly sensitive to thermal contrast and were attracted to stimuli 2.5°C above ambient (Figure 1D–F). Furthermore, mosquito occupancy on the Peltier increased monotonically with stimulus temperatures up to 40°C. However, for higher temperature stimuli, we observed a dramatic reduction in Peltier occupancy. A 50°C stimulus resulted in approximately half as many animals on the Peltier compared to a 40°C stimulus (Figure 1D–F). Stimuli of 55°C or greater resulted in occupancy rates indistinguishable from an ambient thermal stimulus (26°C) (Figure 1D–F). Spatial analysis of mosquito occupancy on or near the Peltier revealed that while mosquitoes were still attracted to high-temperature stimuli, they populated the area peripheral to the Peltier, and strongly avoided the Peltier itself for stimuli ≥ 55°C (Figure 1F). Female mosquitoes searching for a warm-blooded host may be responding to the absolute temperature of a stimulus or may instead be evaluating relative warmth, defined as the differential between a stimulus and background ambient temperature. To investigate the thermotaxis strategies constituting mosquito heat-seeking behavior, we conducted experiments at three ambient temperatures: 21, 26, and 31°C (Figure 2A–F). We found that Peltier occupancy for stimuli 21–40°C depended on the differential between the Peltier and ambient temperature (Figures 2B,C), rather than the absolute temperature of the Peltier (Figure 2A). For example, at all ambient temperatures tested, a stimulus 5°C above ambient was sufficient to elicit significant heat-seeking, and elicited approximately half as much Peltier occupancy as a stimulus 10°C above ambient. On the other hand, heat-seeking to targets 50–55°C was inhibited at all ambient temperatures tested (Figures 2D,F), despite the fact that the temperature differential varied widely in these situations (Figure 2E). Figure 2 with 1 supplement see all Download asset Open asset Mosquitoes thermotax to relative warmth and avoid both relative cooling and stimuli exceeding host body temperature. (A–F) Heat-seeking at different ambient temperatures (n = 5–6 trials per condition): 21°C (blue), 26°C (gray), 31°C (orange). Data in A, B, D, and E are plotted as mean ± s.e.m. (A,D) Percent of mosquitoes on Peltier during seconds 90–180 of stimuli of indicated temperature, normalized to stimulus 10°C above ambient (A, open circle) or 40°C stimulus (D, open circle). (B,E) Same data as in (A) and (D), respectively, plotted using differential between ambient and Peltier temperature. For each ambient temperature, arrowheads indicate the lowest temperature stimulus found to elicit a significant increase in heat-seeking compared to an ambient temperature stimulus (A,B) or a reduction in heat-seeking compared to a 40°C stimulus (D,E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). For each ambient temperature, linear regressions (A, B, 21°C: 10.6/°C, R2 = 0.98, 26°C: 12/°C, R2 = 0.99, 31°C: 9.5/°C, R2 = 0.97) or variable slope sigmoidal dose–response curves (D, E, 21°C: IC50 = 55.4°C, R2 = 0.87, 26°C: IC50 = 52.5°C, R2 = 0.92, 31°C: IC50 = 50.5°C, R2 = 0.91) are plotted. (C,F) Heat maps showing mean mosquito occupancy on the Peltier (red square) and surrounding area, during seconds 90–180 of each stimulus period. Bold borders indicate stimuli with responses significantly different from an ambient-temperature stimulus (C) in (A,B), or significantly different from a 40°C stimulus (F) in (D,E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). (G,H) Analysis of mosquito responses to cooling from data in (Figure 1D). (G) Mean percent of mosquitoes on Peltier during thermal stimuli 31–40°C. Dashed line indicates the end of the stimulus period. (H) Post-stimulus time at which the percent of mosquitoes on Peltier has decayed to one half of the mean during seconds 90–180 of the stimulus period from (Figure 1E). Each replicate is indicated by a dot, mean ± s.e.m. by lines (NS, not significant; one-way ANOVA with Bonferroni correction). https://doi.org/10.7554/eLife.11750.005 These results show that Ae. aegypti thermotaxis is driven by seeking relative warmth, but restricted by an absolute upper threshold of ~50–55°C. Because female mosquitoes are attracted to relative warmth, we hypothesized that they may also avoid relative cool. This complementary behavior would serve to improve host-seeking thermotaxis. We examined mosquito responses to cooling by analyzing the rate at which animals left the Peltier when it cooled at the conclusion of a stimulus period. We found that mosquitoes left the Peltier at similar rates regardless of the absolute temperature of the stimulus (Figure 2G,H, Figure 2—figure supplement 1; based on analysis of data in Figure 1D,E), demonstrating that mosquitoes avoid relative cool during heat-seeking. Our characterization of Ae. aegypti heat-seeking revealed multiple behavioral components contributing to selective thermotaxis during host-seeking: 1) the seeking of relative warmth; 2) the avoidance of relative cool; and 3) the avoidance of thermal stimuli exceeding host temperature. Each of these sensory-motor functions may rely on the same molecular thermosensors, or may instead use distinct thermosensors. We considered the possibility that thermoreceptors ordinarily dedicated to the behavioral thermoregulation typical of most ectotherms such as D. melanogaster, may have evolved a function in host-seeking by mosquitoes and other hematophagous arthropods. Using this reasoning, we generated AaegTRPA1-/- mutants using zinc-finger nuclease-mediated genome editing (Figure 3A, Figure 3—figure supplement 1A). Figure 3 with 2 supplements see all Download asset Open asset AaegTRPA1-/- mutants fail to avoid a chemical irritant and high-temperature stimuli. (A) Representative bright field (left) and fluorescence (right) images of wild-type and AaegTRPA1-/- female pupae marked by ubiquitous expression of enhanced cyan fluorescent protein (ECFP). Scale bars: 0.5 mm. (B) Schematic of capillary feeding (CAFE) assay. (C,D) Sucrose preference over sucrose containing the indicated concentration of N-methylmaleimide (C, n = 10–12 trials per condition) or denatonium benzoate (D, n = 7 trials per condition) for mosquitoes of the indicated genotypes (NS, not significant; *p < 0.05, **p < 0.01; one-way ANOVA with Bonferroni correction compared to wild-type). (E) Percent of mosquitoes of indicated genotypes on Peltier during seconds 90–180 of stimuli of indicated temperature (mean ± s.e.m., n = 6–9 trials per genotype; ***p < 0.001; repeated measures one-way ANOVA with Bonferroni correction). (F) Heat maps showing mean mosquito occupancy for the indicated genotypes on the Peltier (red square) and surrounding area, during seconds 90–180 of each stimulus period. Bold borders indicate stimuli with responses significantly different from wild-type in (E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). (G) Mean percent of mosquitoes of indicated genotypes on Peltier during seconds 0–180 of thermal stimuli 40–60°C and during subsequent re-presentation of 40°C. Timespans with statistically significant increases in AaegTRPA1-/- mutant Peltier occupancy compared to wild-type are indicated by purple lines (calculated from 15 second bins; p < 0.05; one-way ANOVA with Bonferroni correction). https://doi.org/10.7554/eLife.11750.007 In addition to its function as a thermoreceptor, TRPA1 is a highly conserved chemosensor of electrophile irritants such as N-methylmaleimide (Macpherson et al., 2007; Kang et al., 2010). Using a modified capillary feeding (CAFE) assay (Ja et al., 2007) (Figure 3B), we found that wild-type Ae. aegypti mosquitoes strongly avoided consumption of N-methylmaleimide (Figure 3C), as well as the bitter compound denatonium benzoate (Figure 3D). AaegTRPA1-/- mutants rejected denatonium benzoate (Figure 3D) but did not avoid consumption of N-methylmaleimide (Figure 3C). We interpret this result as a loss of N-methylmaleimide detection in AaegTRPA1-/- mutants, leading to no preference between sucrose and sucrose containing N-methylmaleimide. We note that this simple CAFE assay could be used to discover additional mosquito anti-feedants to repel Ae. aegypti, beyond the two chemicals identified here. Because TRPA1 is important in insect thermoregulation (Hamada et al., 2008), we used a modified thermal gradient assay (Sayeed and Benzer, 1996; Hamada et al., 2008) to assess thermal preference in wild-type and AaegTRPA1-/- mutant mosquitoes (Figure 3—figure supplement 1B). AaegTRPA1-/- mutants were impaired in avoidance of high air temperature, leading to significant mortality (Figure 3—figure supplement 1C–G). Together, these data indicate that TRPA1 has a conserved chemosensory and thermosensory function in Ae. aegypti. We next asked if AaegTRPA1 is required for mosquito heat-seeking behavior. AaegTRPA1-/- mutants showed normal attraction to stimuli at or but lacked normal avoidance of higher temperature stimuli and (Figures A analysis of Peltier occupancy over time revealed that AaegTRPA1-/- mutants on the Peltier during and 60°C stimulus control animals rapidly left these high-temperature stimuli (Figure Therefore, AaegTRPA1 is not required for attraction to warmth but is required for normal avoidance of high-temperature stimuli that host body temperature. Because AaegTRPA1-/- mutants attraction to warm stimuli, we used to test a for the Ae. aegypti ortholog of Gr28b, in heat-seeking (Figure 3—figure supplement this is required for rapid avoidance of warmth in D. melanogaster (Ni et al., mutant mosquitoes showed no thermotaxis (Figure 3—figure supplement different of these mutations in Ae. aegypti and D. melanogaster may differences in expression of these is expressed in internal thermosensors of the (Hamada et al., 2008), while thermosensitive of are expressed in peripheral (Ni et al., 2013). In Ae. aegypti, TRPA1 is expressed in including the antennae et al., it may be with peripheral thermosensitive sensilla responding to rapid thermal (Gingl et al., as it is in An. gambiae (Wang et al., 2009). expression of in Ae. aegypti is not but its is expressed et al., 2015). AaegTRPA1-/- mutants did not show normal avoidance of high-temperature stimuli, they may still prefer host-temperature stimuli if with a In a heat-seeking assay with two we examined the of AaegTRPA1 in mosquito thermotaxis in a more complex thermal landscape (Figure In this assay, mosquitoes were with two thermal stimuli. When with two 40°C stimuli, both wild-type and mutant mosquitoes between the (Figure but in a between a 40°C and 50°C wild-type mosquitoes strongly preferred the 40°C Peltier (Figure and avoided the 50°C Peltier (Figure in this AaegTRPA1-/- mutants failed to avoid the 50°C Peltier, resulting in no preference for the 40°C stimulus (Figure D, 1). Figure Download asset Open asset AaegTRPA1-/- mutants fail to discriminate between host-temperature and (A) Schematic of heat-seeking assay. for 40°C 40°C (B) or 50°C 40°C (C) for indicated genotypes (n = 6 trials per genotype; mean ± s.e.m., with each replicate indicated by a not significant; ***p < 0.001; one sample In (C) AaegTRPA1-/- mutants are significantly different from wild-type and mutants (p < 0.05, one-way ANOVA with Bonferroni correction). Heat maps showing mean mosquito occupancy for the indicated genotypes on (red of the indicated temperatures and surrounding area, during seconds of each stimulus period. (E) of mosquito thermotaxis. (F) image of a on a of in in 1 Download asset This be in because may still the for Download as Download as Download as AaegTRPA1 is required for tuning avoidance of high-temperature stimuli during heat-seeking. AaegTRPA1-/- mutants with a between two one at 40°C and one at is up at 1 and at and seconds of the stimulus period. Discussion We have the basic thermotaxis strategies used by and revealed an important role for TRPA1 in this behavior (Figure Using a quantitative thermotaxis assay, we Ae. aegypti heat-seeking behavior in the We found that mosquitoes can for hosts in a range of ambient temperatures by seeking relative warmth and avoiding relative cool. these animals can detect a stimulus with thermal contrast as as In an hosts are warmer than the surrounding air but cooler than sun-warmed and objects (Figure For this mosquitoes such as Ae. aegypti are by to the A more is to for biologically relevant stimuli, and to avoid thermal stimuli exceeding host temperature, as we have observed in our laboratory of heat-seeking. a blood meal is an essential component of for a female Ae. aegypti mosquito. To her of a female mosquito stimuli that host temperatures. Our results that AaegTRPA1 is critical for this selective thermotaxis. heat-seeking behavior an model for of the et al., et al., et al., 2015; et al., and et al., underlying and thermotaxis. of have been restricted to laboratory model such as and Drosophila melanogaster thermotaxis of from thermal environments. Mosquitoes must such behavioral as we have found in our thermal gradient assay. However, their of behaviors is by the of a specialized and highly mode of thermotaxis to locate warm-blooded hosts. It may also be that these two and in mosquitoes. For instance, the avoidance of high-temperature stimuli during heat-seeking may be by a or response of host-seeking behavior. It will be to investigate the neural mechanisms that regulate behavioral of thermoregulation and heat-seeking, and whether these systems are in behavioral during mosquito host-seeking. Our work identifies TRPA1 as a gene mosquito avoidance of high-temperature stimuli, which we have to be a major behavioral component of heat-seeking. However, because both AaegTRPA1-/- and mutants normal attraction to warmth, this of heat-seeking must rely on other thermoreceptors, still to be Our that this attraction must be by a that to background temperature, or multiple thermosensors each to a distinct absolute During with a warm-blooded host or a 50°C Peltier, mosquitoes are likely a range of thermal For instance, there can be a 10°C thermal gradient in the air of a stimulus or human (van Breugel et al., 2015). This that for a mosquito on a warmed Peltier, the brain, and may all be different temperatures. temperature of mosquito will also be by their and as well as thermal to with the Furthermore, in the air near a can be highly and in their with thermal air considerably at the bottom and top of the 2013). These of thermal stimuli may mosquitoes in our assay to be attracted to the bottom and top of the this complex