We created optogenetic tools in A. aegypti mosquitoes that allowed us to precisely activate sensory neurons that are specialized to detect CO₂. To do this we generated a transgenic strain that expresses the red light-activated cation channel CsChrimson translationally fused to the tdTomato fluorescent reporter (Klapoetke et al., 2014) under control of the QF2/QUAS bipartite transcription system (Potter et al., 2010). We crossed this transgene into a strain that expresses the QF2 transcription factor in neurons that express the Gr3 CO₂ receptor subunit (McMeniman et al., 2014; Younger et al., 2022; Figure 1B). CsChrimson-tdTomato was detected in maxillary palp neurons but not antennal neurons, consistent with the observation that maxillary palp neurons are exquisitely sensitive to CO₂ (Grant et al., 1995; Figure 1C and D, Figure 1—figure supplement 1A). As expected, we found that CsChrimson-expressing neurons extended axons that innervated glomerulus MD1 in the antennal lobe of the mosquito brain (Figure 1E), which is known to be CO₂-sensitive (Younger et al., 2020). To test whether mosquitoes responded to optogenetic activation of CO₂ sensory neurons, we presented animals with a 5 s red light (627 nm) stimulus and tracked their movement (Figure 1F–I). Control animals carrying only the Gr3-QF2 driver or the QUAS-CsChrimson transgene reporter showed no response to red light. However, mosquitoes with both genetic elements (Gr3 > CsChrimson) increased their velocity in response to the stimulus (Figure 1H–I). The proportion of mosquitoes responding increased with light intensity (Figure 1—figure supplement 1B-D). The observed increase in activity is consistent with the known role of CO₂ in activating mosquitoes.

When combined with another host cue such as heat, CO₂ is sufficient to elicit blood feeding in female mosquitoes (McMeniman et al., 2014). To test whether fictive CO₂ sensation triggered by optogenetic activation of Gr3 sensory neurons could replace real CO₂, we created a behavior assay called the opto-membrane feeder (Figure 1J). This assay consists of a cylindrical canister of mosquitoes surrounded by red light-emitting diodes (LEDs) and a warm blood meal behind a membrane sitting on top of the mesh lid. Mosquitoes were presented with either CO₂, fictive CO₂ via red light, or neither stimulus. Control mosquitoes with either Gr3-QF2 or QUAS-CsChrimson were attracted to the warm blood meal and engorged only when presented with CO₂ but not when presented with red light (Figure 1K–M). In contrast, Gr3 > CsChrimson mosquitoes were attracted and engorged in the presence of either real or fictive CO₂, the latter delivered as a red light stimulus (Figure 1K–M). These results demonstrate that mosquitoes interpreted optogenetic activation of the CO₂ sensory neurons as a host cue that is sufficient to drive blood feeding.

Host seeking begins when female mosquitoes detect a human, typically by sensing volatile cues like CO₂. Once activated by human odorants, they seek out the source of the cues, and upon landing, mosquitoes walk to locate a patch of skin to pierce (Liu and Vosshall, 2019; McMeniman et al., 2014; van Breugel et al., 2015). To understand the timing and nature of the mosquito response to transient host cues, we created an assay called the opto-thermocycler (Figure 2A–B). In this assay mosquitoes receive optogenetic light stimulation from above and heat through the mesh at the bottom of the assay chamber. The use of fictive CO₂ delivered optogenetically was critical for studying the internal state of the mosquito after these transient host cues. Delivery and removal of real CO₂ necessitates constant air flow, which is itself an important sensory cue for insects (Alvarez-Salvado et al., 2018; Kadakia et al., 2021; Suver et al., 2019).

We employed machine learning-based behavior classification as a high-throughput readout of mosquito behavioral responses (Figure 2C–D). We tracked nine points on the mosquito body using Animal Part Tracker (APT) and four behaviors using Janelia Automatic Animal Behavior Annotator (JAABA) (Kabra et al., 2013): grooming, flying, walking, and probing, a behavior in which the mosquito inserts its proboscis through the mesh in the bottom of the container. When none of these four behaviors were present, mosquitoes were predominantly motionless, showed slow hind leg movement, or occasionally flailed against the wall of the assay chamber without walking. All classifiers showed high accuracy with >90% true positive and true negative rates on a set of test video frames (Figure 2—source data 1).

We delivered 5 s red light pulses and heat increases to simulate brief CO₂ and human body heat stimuli (Figure 2B). Mosquitoes responded to individual heat and fictive CO₂ stimuli with elevated walking, flying, and probing (Figure 2E–H, Figure 2—figure supplement 1). The response to heat alone was dominated by probing and returned to baseline after about 1 min (t_{1/2 probing} = 0.4 min). In contrast, fictive CO₂ alone caused an immediate flight and probing response followed by sustained walking, flying, and probing (Figure 2H, Figure 2—figure supplement 1, Video 1) that took approximately 15 min to return to baseline (t_{1/2 probing} = 3.9 min). Varying the light intensity changed the proportion of mosquitoes responding but not the duration of the response (Figure 1—figure supplement 1B-D). The long duration of the response to CO₂ is reminiscent of persistent internal states in other organisms (Asahina et al., 2014; Flavell et al., 2013; Kohatsu et al., 2011; Hindmarsh Sten et al., 2021; Marques et al., 2020).

These observations of mosquito behavior could reflect an internal state specific to host seeking or a general arousal state elicited by many sensory stimuli. Like CO₂, bright light is also an arousal signal in mosquitoes (Araripe et al., 2018) so we asked whether bright light induces a long-lasting behavior state. Mosquitoes have weaker visual sensitivity to red wavelengths (San Alberto et al., 2021), and we saw no behavioral response to red light (Figure 2E), so we used green light. Because this experiment is designed to test whether mosquitoes have a response to a visual stimulus, a question that does not depend on the use of optogenetics, we used wild-type mosquitoes. A bright green light stimulus induced a brief response dominated by walking (t_{1/2 walking} = 0.4 min), much shorter than the response to fictive CO₂ (Figure 2I and K).

While CO₂ and heat elicit the blood-feeding program required for females to produce eggs, mosquitoes possess a second, distinct feeding program for ingestion of flower nectar for energy (Jové et al., 2020; Lahondère et al., 2020). We asked whether optogenetic stimulation of Gr4 sensory neurons that are thought to detect sugar evoked a sustained behavior response in mosquitoes as with optogenetic activation of the CO₂ sensory neurons. We found that fictive sugar elicited a response composed largely of probing that was of shorter duration than fictive CO₂ (t_{1/2 probing} = 1.5 min) (Figure 2J and L).

The temporal resolution of our assays allowed us to understand precisely how mosquitoes integrate CO₂ and heat to affect their behavior. We focused on the first 15 s after stimulus onset during which the greatest behavior responses are seen. We found that the heightened probing response seen when the stimuli were presented together was roughly additive with respect to the individual stimuli (Figure 2M, Figure 2—figure supplement 2). In contrast, flying was strongly suppressed. This demonstrates that multimodal integration of host cues biases action selection away from long-range flight and toward probing, a short-range behavior.

Heat is a host cue but also may be experienced by the mosquito under other environmental contexts. We compared the integration of heat with green light and fictive sugar to see if they are integrated similarly to the host cue CO₂. In contrast to the integration of host cues, probing was suppressed when non-host cues were presented together (Figure 2M, Figure 2—figure supplement 2). This demonstrates that the mosquito nervous system uses different computations for the integration of host cues and non-host cues.

The fact that bright green light and fictive sugar stimuli elicited briefer responses suggested that the prolonged response to fictive CO₂ is specific to host seeking. If true, it should be modulated similarly to host-seeking behavior. Host seeking in A. aegypti is suppressed after a female takes a blood meal, only returning after she lays eggs several days later (Duvall et al., 2017; Klowden, 1981). We allowed female mosquitoes to blood feed on a human arm and then assayed their behavior 4 days later. We found that blood-fed females completely lost their response to fictive CO₂ and nearly completely lost their response to heat (Figure 3A, B, D and E). For comparison, we asked whether blood-fed females could respond to the optogenetic sugar stimulus by activating Gr4 sensory neurons. We found that blood-fed females had reduced responses to fictive sugar, but unlike fictive CO₂, the response was still detectable (Figure 3—figure supplement 1A-E). This demonstrates that blood-fed females specifically show a complete loss of the persistent state elicited by fictive CO₂.

Unlike females, male mosquitoes do not seek out hosts to feed on blood. Males do demonstrate a flight response to CO₂ (Matthews et al., 2016) and are reported to congregate in the vicinity of humans where they mate with female mosquitoes (Hartberg, 1971). We observed that male mosquitoes had minimal responses to heat, but substantial flight and walking responses to fictive CO₂ (Figure 3C–E). However, the response to fictive CO₂ was brief, decaying rapidly back to baseline (t_{1/2 probing} = 0.4 min). This observation suggests that the persistence—but not the initial response—is specifically regulated in a sexually dimorphic manner.

We have previously shown that fruitless mutant male mosquitoes gain strong attraction to human odor (Basrur et al., 2020). We asked whether these fruitless mutant males have an altered response to fictive CO₂ stimuli. First, we confirmed that fruitless mutant males lacking Gr3 > CsChrimson did not respond to red light, as expected (Figure 3F). fruitless heterozygotes receiving fictive CO₂ showed a brief response (t_{1/2 probing} = 0.4 min) (Figure 3G1 and J), similar to the response we saw for wild-type males (Figure 3C). By comparison, fruitless mutant males receiving fictive CO₂ showed a strong and sustained response for minutes after the stimulus (t_{1/2 probing} = 1.7 min) (Figure 3H–J). This suggests that fruitless is involved in the regulation of the sexual dimorphism of the persistent host-seeking state. We note that the duration of the sustained response of fruitless mutant males is shorter than in females, suggesting additional sexually dimorphic factors may regulate this internal state. Taken together, these results demonstrate that blood-fed females and males, which do not engage in blood-feeding behavior, lack sustained responses to brief pulses of fictive CO₂. This raises the possibility that the persistent state is the behavioral mechanism by which the goal of blood feeding is sustained in the female mosquito.

Motivation consists of two components: increased arousal and directed action toward a goal. We have demonstrated that fictive CO₂ induces a prolonged increase in movement and the probing behavior that immediately precedes blood feeding. We asked whether the persistent state induced by a brief pulse of fictive CO₂ can influence the response to body heat and ultimately if it can induce blood feeding many minutes afterward.

First, we tested whether fictive CO₂ primes subsequent responses to heat (Figure 4A). Because CO₂ is highly volatile, mosquitoes likely sense this cue before body heat in naturalistic host-seeking settings. When we presented heat first followed by fictive CO₂, relatively small behavioral responses were evoked (Figure 4B–C). However, when fictive CO₂ was presented simultaneously with heat or 15 or 60 s prior to heat, larger walking and probing responses were seen (Figure 4B–C). This suggests that mosquitoes respond most strongly to the naturalistic temporal order of these host cues. Next, we asked how long prior to the heat stimulus fictive CO₂ can boost the response (Figure 4D). Compared to heat alone, walking and probing were detectably increased when a fictive CO₂ stimulus was presented up to 4 min prior (Figure 4E–F). This demonstrates that in addition to the sustained behavior response, fictive CO₂ increases the response to heat for minutes afterward.

Once a mosquito pierces the skin of a human host, taste cues present in the blood guide the decision to engorge. Because this is the final goal of host-seeking behavior, we wondered whether mosquitoes in the fictive CO₂-triggered persistent state had altered responses to both heat and taste stimuli. To test this, we designed a blood blanket assay that incorporated a thin sheet of a blood meal mimic located between the thermocycler heating element and the mesh below the mosquito, allowing it to be rapidly heated and cooled (Figure 4G–H). We used a solution of ATP in saline, which has previously been shown to be a highly palatable meal that induces females to engorge in the same manner as blood (Galun et al., 1963; Jové et al., 2020). This allowed us to test how the prolonged arousal state induced by fictive CO₂ influences the decision of the mosquito to feed on a blood meal mimic.

First, we explored the temporal relationship of host stimuli to test whether the naturalistic order of CO₂, heat, and taste stimuli elicits greater rates of feeding. When fictive CO₂ was presented along with heating of the meal for 10 min, many females fed to repletion (Figure 4I). To test the temporal order of these cues, we offered fictive CO₂ and a meal that was only warmed for several minutes, resulting in reduced feeding levels (Figure 4I). When the order was swapped and females were briefly offered the warm meal prior to fictive CO₂, very few females fed (Figure 4I). This suggests that mosquitoes feed at the highest rates when they receive a CO₂ stimulus prior to heat and taste stimuli.

Next, we asked how long after a brief pulse of fictive CO₂ females would retain the motivation to feed on the blood meal mimic. When offered the warm meal without fictive CO₂, few females fed. We then stimulated the females with fictive CO₂ and heated the blood meal mimic either immediately or after a delay of 2, 8, 14, or 20 min after fictive CO₂. We hypothesized that if the fictive CO₂ induced a persistent state of host seeking, it might trigger engorging behavior many minutes later. Indeed, fictive CO₂ was able to potentiate feeding when presented up to 14 min prior to heating of the blood meal mimic (Figure 4J). Taken together, our results demonstrate that the persistent host-seeking state increases host-seeking behaviors and alters the response to sensory cues for many minutes after a brief fictive CO₂ stimulus. We speculate that this reflects the amount of time a mosquito will pursue a host in a naturalistic setting before halting the search if it appears that the host is no longer nearby.

We noticed that there is considerable individual variation in how mosquitoes respond to the warm blood meal mimic after being activated by fictive CO₂ (Figure 4J). We asked whether the behavior state of individual mosquitoes could be inferred over longer periods of time. We extracted 38 behavior parameters from the experiment in Figure 4J from 30-s-long time windows and used t-distributed stochastic neighbor embedding (tSNE) to visualize the relationships. This embedding revealed that mosquito behavior fell into four major states that we termed rest, global search, local search, and engorge (Figure 4K, Figure 4—figure supplement 1). These states were observed even when the length of the time window was varied over a sixfold range (Figure 4—figure supplement 2). The states differed in the proportions of behaviors observed, the transition rates between them, and the total distance the mosquito travelled while in each state. We divided the data into those females that eventually fed or did not feed at the end of the experiment (Figure 4L). As expected, the engorge state was greatly enriched in mosquitoes that were categorized as fed, demonstrating that this method is effective at identifying longer-timescale behavior states (Figure 4L).

We asked whether the behavior state of the mosquito after fictive CO₂ was associated with the future decision to feed on the warm blood meal mimic. We found that fed and unfed mosquitoes showed similar amounts of global search pre-heat but greater levels of local search for those that would later feed (Figure 4M). The local search state differs primarily by showing more probing and less flight than the global search state. To avoid potential distortions created by the two-dimensional embedding (Chari et al., 2021), we trained a linear classifier using only the four behaviors (groom, walk, flight, and probe). A classifier trained on the proportion of time mosquitoes spent in each behavior for 2 min after the light stimulus could predict which mosquitoes would later feed with above-chance accuracy (Figure 4N). Thus, the behavior state that individual mosquitoes enter in response to fictive CO₂ reflects the likelihood of response to future sensory cues.

The following A. aegypti strains were used in this paper: wild-type Liverpool, Gr3-QF2 (Younger et al., 2022), Gr4-QF2 (Jové et al., 2020), fruitless^∆M (Basrur et al., 2020), fruitless^{∆M-tdTomato} (Basrur et al., 2020), and QUAS-CsChrimson (this study).

Mosquito strains were reared at 26°C ± 2°C with 80% humidity and 14 hr light, 10 hr dark (lights on at 7 AM) as previously described (DeGennaro et al., 2013). Embryos were hatched in hatching broth: 1 pellet of fish food (TetraMin Tropical Tablets, Pet Mountain 16110M) crushed using a mortar and pestle, added to 850 mL deionized water, then autoclaved. Larvae were reared in deionized water and fed 1–3 tablets of fish food per day. Adult mosquitoes were fed on 10% sucrose (w/v in distilled water) ad libitum. Sucrose was delivered in a Boston clear round 60 mL glass bottle (Fisher FB02911944) filled with 50 mL 10% sucrose. A cotton dental wick (Richmond Dental 201205) was inserted into the bottle and mosquitoes fed from the sugar-moistened wick. Female mosquitoes were blood fed on mice or human arm to generate eggs. Eggs were dried at 26°C and 80% humidity for 3 days, and then stored at ambient temperature and humidity for up to 3 months. Adults were allowed to mate freely for at least 7 days prior to performing experiments. All behavior experiments were carried out in the light phase of the photoperiod, with most experiments occurring between zeitgeber (ZT) ZT2 and ZT12.

We generated mosquitoes that expressed a translational fusion of CsChrimson to tdTomato under the control of the QUAS promoter, referred to as CsChrimson or QUAS-CsChrimson throughout the paper. The coding sequence of CsChrimson-tdTomato was PCR-amplified from the vector p20X (Klapoetke et al., 2014) using the following oligonucleotide primers: forward 5’- CTCGAGCAAAATGAGCAGACTGGTCGCCGCTTC-3’, reverse 5’- ATCCTCTAGATTACACCTCGTTCTCGTAGCAGAATTTATACAG-3’. The vector backbone from pXL-BacII-15xQUAS_TATA-SV40 (Riabinina et al., 2016) was amplified by PCR using the following oligonucleotide primers: forward 5’-GTCTGCTCATTTTGCTCGAGCCGCGGCCGCAGATC-3’, reverse 5’-CGAGGTGTAATCTAGAGGATCTTTGTGAAGGAACCTTACTTCTG-3’. The CsChrimson insert was cloned into the backbone using Infusion HD cloning kit (Takara 638920) to create pTS26, available at Addgene (plasmid number 175548). This plasmid was injected into 500 A. aegypti Liverpool embryos by the Insect Transformation Facility (Rockville, MD) using 200 ng/µL plasmid DNA and 200 ng/µL piggyBac transposase mRNA. Ten independent QUAS-CsChrimson integration events were isolated under standard mosquito rearing conditions.

Mosquitoes 3–4 weeks of age were anesthetized on ice, then maxillary palps and antennae were removed using sharp forceps and placed in fixative (4% paraformaldehyde, 0.1 M Millonig’s Phosphate Buffer pH 7.4, 0.25% Triton X-100) and nutated for 30 min at 4°C. Tissues were washed four times in PBS, then mounted in SlowFade Diamond Antifade Mountant (Thermo Fisher). Images were acquired on an Inverted LSM 780 laser scanning confocal microscope (Zeiss) using a 25 × 0.8 NA multi-immersion objective with oil. Images were processed using ImageJ.

Brain immunostaining was carried out as previously described (Jové et al., 2020). Mosquitoes 1–2 weeks of age were anesthetized on ice, then heads were removed using forceps and placed into fixative (4% paraformaldehyde, 0.1 M Millonig’s Phosphate Buffer pH 7.4, 0.25% Triton X-100) and nutated for 3 hr at 4°C. Heads were washed four times in PBS and kept on ice during dissections. The brains were dissected using #5 forceps (Dumont) in a droplet of PBS on a Petri dish coated with SYLGARD silicone elastomer (Dow). Brains were transferred to a 35 µm mesh cap of a flow cytometry test tube (Fisher 08-771-23) in a 24-well plate containing PBSTx (PBS with 0.25% Triton X-100). Brains were washed four times for 30–60 min at room temperature in PBSTx on an orbital shaker before permeabilization and between each of the following steps. Brains were permeabilized in PBS with 4% Triton X-100% and 2% normal goat serum for 2 days at 4°C on an orbital shaker. We used the mouse anti-Bruchpilot (brp) monoclonal antibody at a dilution of 1:10,000. The brp antibody was purified by Frances Weis-Garcia of the Sloan Kettering Institute Antibody & Bioresource from the brp/nc82 hybridoma, developed by Erich Buchner at the Universitätsklinikum Würzburg and obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA. Rabbit anti-RFP antibody (Rockland 600-401-379) was used at a dilution of 1:1000 to detect tdTomato fused to CsChrimson as well as the dsRed transgene marker expressed from the 3XP3 enhancer/promoter. Brains were incubated in primary antibodies in PBSTx with 2% normal goat serum for 3 days at 4°C on an orbital shaker. Secondary antibodies were goat anti-mouse Alexa Fluor 647 (Thermo Fisher A21235) and goat anti-rabbit Alexa Fluor 555 (Thermo Fisher A32732) both at 1:500 dilution. Brains were incubated in secondary antibodies in PBSTx and 2% normal goat serum for 2 days at 4°C on an orbital shaker. Brains were washed four more times at room temperature for 30–60 min before mounting in SlowFade Diamond Antifade Mountant (Thermo Fisher). Images were acquired on an Inverted LSM 880 Airyscan NLO laser scanning confocal microscope (Zeiss) using a 25 × 0.8 NA multi-immersion objective with oil. Images were processed using ImageJ.

For CsChrimson to respond to red light (625 nm in this paper), it is necessary to supply the all-trans retinal co-factor. Moreover, it is critical that animals reared for optogenetics be maintained in the dark to avoid activating CsChrimson inappropriately. Therefore, we developed a mosquito rearing protocol to deliver all-trans retinal under dark conditions. First, we carried out experiments to select the best QUAS-CsChrimson transgenic insertion among the 10 lines we generated. Insertions were identified and tracked using fluorescence from the 3xP3-ECFP marker. We wanted lines with strong and selective behavioral induction in combination with a QF2 driver, but no basal behavioral activity without a QF2 driver. We also wanted it to be a single insertion at a known position in the genome, that would not obviously disrupt a known gene. The insertion site of the transgene in each line was mapped to the genome using TagMapping (Stern, 2016). After being fed with all-trans retinal as described below, all 10 lines were tested for their response to red light with and without being crossed to a QF2 driver. Based on these initial screens, a single line with the QUAS-CsChrimson transgene inserted in an intron of the gene LOC23687794 on chromosome 2 at base pair 453,953,698 in the L5.0 version of the A. aegypti genome (Matthews et al., 2018) was selected for use in all subsequent experiments. This CsChrimson strain was outcrossed to wild-type mosquitoes for eight generations before being homozygosed and used for behavior experiments. For all experiments except those in Figure 2I and K, which used wild-type mosquitoes with a green light startle stimulus, animals were subjected to special rearing conditions to prepare them for optogenetics. Eggs were hatched in 1 L of hatching broth under a 14 hr 450 nm blue light and a 10 hr dark cycle in a light-tight 26°C incubator with 80% humidity. Blue light was selected for the light phase of the photoperiod to avoid activating CsChrimson. The next day 2 L of distilled water was added to the pan, and the following day larvae were thinned to 450 per pan. Larvae were subsequently sorted for fluorescence markers if necessary, using a dissecting microscope. Larvae were fed daily with 1–3 tablets of Tetramin fish food (Pet Mountain 16110M) ground into a powder using a mortar and pestle. Pupae were moved to eclose into adults in a 30 cm × 30 cm × 30 cm insect rearing cage (Bugdorm DP1000) with ad libitum access to 10% sucrose in sugar-feeding glass bottles. Animals were not sexed at this stage, so cages contained males and females that freely mated. Behavior experiments were performed 1–4 weeks post-pupation. Three days before the experiment, the sugar wicks were replaced with water wicks to starve animals for 24 hr. Two days before the experiment, the water wicks were replaced with wicks soaked in 10% sucrose and 400 µM all-trans retinal (Sigma, R2500-1G). Fifty mL of sucrose and all-trans retinal was used per cage. Animals were allowed to feed for 1–3 days in the dark on this meal. In pilot experiments we verified that starved females fed on sucrose and all-trans retinal by observing yellow pigmentation in the abdomen. Feeding in the dark was used to avoid premature neuronal activation and bleaching of the all-trans retinal in the sugar feeders. This rearing protocol was used for all experiments in the opto-membrane feeder, opto-thermocycler, and blood blanket experiments.

The opto-membrane feeder assay was constructed using optomechanical components (Thorlabs MB12, TR12, RA90) and a black 1/4" thick acrylic platform for the canister of mosquitoes to rest on. A hole in the bottom of the platform allowed a camera (Blackfly U3-13S2M-CS, FLIR) outfitted with an 800 nm longpass filter (Midwest Optical LP800-34) to image through the clear canister. Canisters were constructed from a polycarbonate tube of diameter 4.5" (McMaster-Carr 8585K56) and 5" long. The bottom was made of clear 1/8" thick acrylic and attached with plastic epoxy (Loctite 1363118). The top was an inset lid made of black 1/4" and 1/8" acrylic and UV-resistant black mesh (McMaster-Carr 87655K13). The canister was surrounded by a coil of RGB LEDs (Digikey 289-1189-ND) spaced 1.5" from the exterior of the canister and controlled by an Arduino Uno board (Arduino A000066). Mosquitoes were illuminated by 850 nm infrared LEDs surrounding the top of the cylinder of RGB LEDs. The assay was enclosed in a black 1/4" thick acrylic box of dimensions 15" × 15" × 28" to prevent ambient light from entering the assay. The top of the acrylic box had an entry port of 4" × 2.7" for CO₂ diffused by a Flystuff Flypad (Genesee Scientific, 59-114), and two doors on the side, one at the level of the cylinder (10" high × 8" wide) and one at the bottom (8" high × 10" wide) at the level of the camera. The day before the experiment, mosquitoes were sexed under cold anesthesia in white light, placed into the cylindrical canisters, and fed water and 400 µM all-trans retinal in the dark until the experiment commenced. Dental wicks were soaked in approximately 12.5 mL of the water and all-trans retinal, placed on top of the mesh of the inset lid. Trials were run in an environmental room at 25–28°C and 70–80% humidity. For each trial, a canister of 20 mosquitoes was placed on the platform and acclimated for 10 min prior to the stimulus. Throughout the acclimation period and trial, the canister was bathed in dim blue 471 nm light from the RGB LEDs. The RGB LEDs were arranged in a coil around the cylindrical canister of mosquitoes to give a relatively even light distribution throughout the canister as measured by a light meter (Coherent Wand UV/VIS Power Sensor 1299161). At the start of the trial, a blood meal consisting of 5 mL of defibrinated sheep blood (Hemostat Laboratories DSB100) with 2 mM ATP (Sigma A6419-1G) heated to 42°C was placed on top of the canister. Blood meals were delivered using an acrylic lid consisting of a 1/16" thick clear acrylic ring with a 2" inner diameter and 2.6" outer diameter attached to a 1/2" thick clear acrylic ring with a 2.3" inner diameter and 2.6" outer diameter. This lid was covered with Parafilm on the 1/16" thick side to create a well for the blood when placed Parafilm-down on the inset lid of the cylindrical canister. At the start of the experiment, the warm blood meal was pipetted onto the Parafilm. On top of the blood meal was an inverted 4 oz bottle (SKS Bottle & Packaging 0604–07) filled with water heated to 42°C to keep the blood near body temperature for the duration of the 15 min trial. Mosquitoes were given CO₂, red light (624 nm, 3.5–6 µW/mm²), or neither stimulus throughout the 15 min trial. The light intensity chosen was an intermediate intensity as determined by a light-behavior dose-response curve (Figure 1—figure supplement 1B-D); 10% CO₂ was mixed with filtered room air using flow controllers (Aalborg P26A1-BA2) to deliver a 2.7% CO₂ stimulus through the top of the container. Air flow delivery is described in detail in Basrur et al., 2020. Between trials, the lower door was opened for 5 min with the air flow on to flush residual CO₂ from the assay. On a given day of experiments, each of three genotypes (Gr3, CsChrimson, and Gr3 > CsChrimson) was tested with each of the three stimuli (no stimulus, CO₂, red light), for a total of nine trials. The order of trials was rotated between days. Genotypes were blinded to the experimenter. Attraction to the warm blood meal was quantified by manually counting the number of mosquitoes on the warm blood meal in the video (1 frame/second) every 15 s. Engorgement was quantified by visual examination of mosquitoes at 4°C after the end of the trial. Between days of experiments, the canisters were cleaned by spraying 70% ethanol with a spray bottle and wiping down with a soft sponge, rinsed with deionized water, and air dried.

The opto-thermocycler assay was constructed on top of a PCR thermocycler (Eppendorf Mastercycler) using optomechanical components (Thorlabs XE25L12, XE25L24, XE25L09, XE25T4, RA90, TRA6, TR12). This assay was used as the basis for experiments delivering light only, light along with heat stimuli, and blood blanket experiments. Light was delivered using six red light 627 nm LEDs (Luxeon Star SP-01-D9) or six green light 530 nm LEDs (Luxeon Star SP-01-G4) controlled with an Arduino Uno board. Light intensity was measured using a Coherent Wand UV/VIS Power Sensor (1299161) at nine points on the surface of the PCR block and light angle was adjusted using wires to achieve even illumination. The surface of the PCR block was covered in black tape to reduce glare (Thorlabs T137-2.0). Temperature was measured using a type T thermocouple (Harold G Schaevitz Industries LLC CPTC-120-X-N) connected to the Arduino board (Arduino A000066) using a thermocouple amplifier (Adafruit MAX31856). The thermocouple sensor was placed on the surface of the lower right of the PCR block and secured using black tape (Thorlabs T137-2.0). Temperature reading and light output were recorded every 100 ms from the Arduino using a custom Processing script. Video was synchronized with the light and temperature stimuli with an infrared 940 nm LED (Adafruit 387) covered with tape and placed in the field of view of the camera. Mosquitoes were illuminated with an infrared 850 nm LED strip (Waveform Lighting 7031.85) surrounding the plate of mosquitoes orthogonal to the view of the camera. Video was recorded using a Blackfly camera (FLIR BFS-U3-16S2M-CS) outfitted with a 780 nm longpass filter (Vision Light Tech LP780-25.5) at 30 frames/s using Spinview software. Heat stimuli were programmed onto the PCR thermocycler to elicit the desired change in temperature from ambient to skin temperature (25–35°C) as measured by the thermocouple (Figure 2B). 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 a decrease in the behavioral response. Green light stimuli were 530 nm at an intensity of 22 µW/mm², chosen because it was the maximum intensity of our setup and to maximize the chance of observing a persistent response to green light. To synchronize the heat and light stimuli, experiments started with a brief dip in temperature followed by a 10 min acclimation period after which the experiment started. Experiments in Figure 2E–H were conducted with a single stimulus presented to mosquitoes to determine the duration of response. In all other experiments, mosquitoes received multiple stimuli over the course of a 3–6 hr experiment. Data from the rare trials where the mosquito died during the experiment were discarded. For experiments using red light and heat, trials were delivered 20 min apart and the order was pseudorandomized between multiple sweeps of trials and across days. For experiments using green light and heat (Figure 2I and K), the stimuli were pseudorandomized across sweeps only. Sweeps are considered technical replicates conducted on the same individual mosquito, and each mosquito in the assay is a biological replicate. All experiments were conducted multiple times either as a pilot followed by full experiment or multiple full experiments. Mosquitoes were assayed in a custom acrylic plate with 3 × 5 wells. The sides of the plate were cut using a laser cutter from 1/8" thick clear acrylic, then assembled using acrylic glue (WELD-ON, #4SC Plastic Solvent Glue for Acrylic). The top and bottom were cut from 1/16" acrylic. The top was left removable to load mosquitoes while the bottom was used to sandwich a piece of black fiberglass window screen (Breakthrough Premium Products IHLRS3684BL) creating a mesh bottom for each well. The acrylic bottom piece spaced the mesh bottom of the wells 1.5 mm from the surface of the PCR block. Wells containing the mosquitoes were 18.5 mm long × 17 mm wide × 12 mm high. The well in the lower right was empty to accommodate the thermocouple. The day before the experiment, mosquitoes were sexed under cold anesthesia in white light, placed into the custom plate, and fed water and 400 µM all-trans retinal overnight until the experiment. This was delivered in cotton dental wicks each soaked with 12.5 mL water and all-trans retinal. Three wicks were laid flat beneath each plate so that mosquitoes in all wells could access the wicks beneath. Experiments were run at ambient room temperature and humidity, but the PCR block kept the assay chamber at a fixed temperature. Between trials the surface of the PCR block was cleaned by wiping with a Kimwipe moistened with 70% ethanol. Between days of experiments, the acrylic plates were cleaned by spraying 70% ethanol with a spray bottle and wiping down with a gloved finger, rinsed with deionized water, and air dried.

The opto-thermocycler assay captures probing behavior but does not offer a meal for engorgement. We therefore modified this device to produce the blood blanket assay. The most biologically relevant meal for host-seeking females would be blood, but its opacity makes it unsuitable for our video tracking. We therefore used adenosine 5'-triphosphate (ATP) in saline as a an optically clear proxy for blood. This meal has previously been shown to be highly palatable and triggers mosquito engorgement equivalent to a blood meal (Galun et al., 1963; Jové et al., 2020). To modify the opto-thermocycler to accommodate this blood meal substitute, a thin aluminum plate (McMaster-Carr 6061 Aluminum sheet 0.025") was sandwiched between laser cut pieces of acrylic creating wells on the side facing the mosquito. The wells were 18.5 mm × 17 mm × 1 mm. The acrylic was bonded to itself using acrylic glue (WELD-ON, #4SC Plastic Solvent Glue for Acrylic) and to the aluminum plate with epoxy (Loctite 1363118) and UV-curing glue (Bondic SK8024). The plate was prepared for a trial by adding 500 µL of the meal (110 mM NaCl, 20 mM NaHCO₃, and 2 mM ATP) to each well of the plate in Figure 4A–C. In Figure 4D–F, the composition of the meal was 110 mM NaCl, 10 mM NaHCO₃, and 2 mM ATP. The plate was covered with Parafilm to provide a membrane for the mosquitoes to pierce before accessing the meal. The plate was placed directly on top of the PCR block to allow maximum heat transfer. The thermocouple was placed on the surface of the Parafilm in the middle of the well in the lower right corner to record the temperature of the heated meal. Trials were carried out and synchronized in the same way as opto-thermocycler experiments. All blood blanket experiments were single trial.

Videos were pre-processed using a custom Python script tracking_optothermo.py that converted the file format, split up videos into ~30 min chunks, selected frames to create a background image for centroid tracking, and detected frames where the IR synchronization LED was illuminated. Next, we used Ctrax (Branson et al., 2009) for centroid tracking. A background model was created using the selected frames from the experimental video. Ctrax background settings were background brightness high threshold 2.55, low threshold 0.25–0.5 adjusted depending on the video. The area with the infrared synchronization LED was excluded using a region of interest to avoid interference with the tracking. Mosquitoes that moved very little, such that they were visible in the background image, were corrected for using the Fix Background Model option. In tracking settings, shapes were filtered using the following minimum/maximum: 110/1600 for area, 4/36 for major axis, 4/30 for minor axis, 0.0/0.98 for eccentricity. The rest of the tracking settings were default. We used Ctrax centroid tracking as input to APT (https://github.com/kristinbranson/APT downloaded on July 9, 2020; Branson, 2022) for tracking points on the mosquito body. For opto-thermocycler experiments, we tracked nine points: the tip and base of the proboscis, the tip of the abdomen, and three points on each foreleg: where the femur connects to the body, the joint between the femur and the tibia, and the joint between the tibia and the first tarsomere. Tracking the mid legs and hind legs was not needed for behavioral classification so they were excluded to speed computation. Opto-thermocycler classifiers were trained on 320 frames from two videos for female mosquitoes and 102 frames from one video for male mosquitoes. We tracked 13 points for blood blanket experiments, the same nine points as for opto-thermocycler experiments plus two points at the point of the abdomen where it connects to the thorax and two points at the midpoint or thickest part of the abdomen. The blood blanket classifier was trained on 215 frames from four videos. All APT classifiers were trained using the Cascaded Pose Regression tracking algorithm. JAABA (Kabra et al., 2013) (downloaded on July 15, 2020) was used for classifying specific behaviors. The classifier for flight (called fly2) was used for all videos of females and males. It was trained from two videos and used appearance and locomotion features with radius of 10 frames with no post-processing. The other classifiers additionally used APT information, a larger radius of frames, and minimum bout sizes for improved accuracy. Separate classifiers were trained for females and males in the opto-thermocycler and females in the blood blanket experiments to maximize classifier accuracy in the face of differences in visual appearance. Probing classifiers (probe5 for female opto-thermocycler experiments, probemale for male opto-thermocycler experiments, and probeBB for females in the blood blanket experiments) included the pair of points proboscis tip and base as features, along with APT, motion, and appearance features. The grooming and walking classifiers (walk3 for female and male opto-thermocycler experiments, groom3 and groommale for female and male opto-thermocycler experiments, respectively, and walkBB and groomBB for blood blanket experiments) were trained using APT, locomotor, and appearance features. APT classifiers were visually inspected for accuracy. APT and JAABA classifiers were evaluated by the accuracy of ground truthing on the JAABA classifiers. An initial classifier was trained, then ground truthing was performed on 50–100 segments of 1 s video segments that were balanced between segments with and without the behavior. These segments were examined for mis-classified frames and additional training was performed to improve the classifier. Thus, the ground truth dataset is more challenging than a random one because it contains frames that were previously mis-classified and so the real accuracy is higher. Training continued until true positive and true negative rates of >90% were obtained with seven of nine classifiers. Two other classifiers had rates slightly below this. The groomBB was trained to ~84% true positive and negative rates because certain grooming postures are difficult to distinguish from probing postures. The probemale classifier was trained to ~87% true positive and ~91% true negative rates because only part of the male proboscis is distinguishable from the maxillary palps during probing behavior. Classified behaviors for each mosquito track from JAABA were assigned to single wells according to x–y location of the track to correct the small numbers of frames where Ctrax detected two mosquitoes per well (usually due to a leg that was discontinuous with the rest of the animal) and to connect broken tracks to a single individual mosquito. The IR LED stimulus in the video was aligned with data about temperature and light stimuli from the Arduino and assigned to frames in the video. Velocity was calculated by taking the Ctrax x–y position at 100 ms intervals (three frames).

To calculate the half-life of the mosquito behavior response in Figures 2H–J–3C and H, the baseline was calculated as the average probing in 2 min prior to stimulus onset. A sliding window of the amount of probing was calculated in 15 s windows starting at stimulus onset for every frame. The maximum response was defined as the window with the greatest probing after stimulus onset and t_1/2 was defined as the first window in which the probing was halfway between the maximum response and the baseline. To calculate the integration of heat and the second stimuli (fictive CO₂, fictive sugar, or green light) in Figure 2—figure supplement 2 and Figure 2M, we calculated the average response to each of the individual stimuli. We added the two responses to get a predicted additive response. For each individual mosquito, we divided its response by the predicted additive response and multiplied by 100%. This gave a percent additivity where 0% was no response and 100% was exactly additive. For line graphs, the additivity signal was smoothed over 4.5 s around each 500 ms timepoint.

To infer the state of individual mosquitoes in the blood blanket experiment, we split each mosquito track into 30 s intervals at 10 s step size and calculated 38 parameters. The 30 s time interval was selected as a period of time over which the behaviors exhibited were relevant to interpreting the internal state of the mosquito. The time interval was varied from 10 to 60 s to assure that the results were not sensitive to this parameter choice (Figure 4—figure supplement 2). The parameters included the proportion of the time window that mosquitoes exhibited each behavior and no behavior. Mosquitoes can probe and walk at the same time so the proportion of time probing and walking, probing not walking, and walking not probing were included. The number of bouts of each behavior was included. Velocity parameters included average velocity over the window and average velocity during each behavior. Transitions between behaviors were included as outgoing rate per second of transition to all other behaviors or no behavior. For the purposes of transitions and ethograms, probing and walking were treated as mutually exclusive with probing taking higher precedence over walking. For all behaviors to avoid rare frames where multiple behaviors were classified for a single frame, the precedence of behaviors were flying > probing > walking > grooming > no behavior. Based on the total amount of time animals spent performing each behavior, cutoffs were determined to specify a minimum amount of behavior exhibited. Cutoffs were 0.04 for flight, 0.2 for walking or probing, and 0.3 for grooming. Behavior below these cutoffs was excluded from further analysis. The Python package scikit-learn (https://scikit-learn.org/ version 0.24.1) was used for tSNE with parameters n/100 perplexity (1061), and other default parameters (200 learning rate, 1000 iterations). Multiple perplexities were compared to assure that results were not sensitive to this parameter choice. tSNE plots were examined and clusters were segmented manually by grouping densely clustered points. These clusters were used to annotate videos for visual inspection of what mosquito behaviors they corresponded to. Names for clusters were chosen based on the characteristics of the clusters shown in Figure 4K, Figure 4—figure supplement 1B, Figure 4—figure supplement 2, and video observation. Clusters that included mosquitoes that moved around were named Global or Local search based on the total amount of movement and contrasting amounts of flight and walk behaviors. The cluster that included mostly grooming was termed Rest. The cluster that included mosquitoes that were stationary, probing, and with abdomens expanded from feeding was termed Engorge. The clusters for Rest, Global Search, and Local Search were single clusters that were clearly differentiated on the tSNE. The Engorge cluster was composed of two smaller clusters that, when observed on video, both consisted of mosquitoes engorging and were therefore combined. Points on the end of the Local Search cluster in the tSNE with high probing were also examined by video and grouping was kept with the Local Search cluster.

R (https://www.r-project.org version 4.0.5) and Python were used for statistical analysis. Data distributions were visually examined for normality or tested using the Shapiro-Wilk test. Normally distributed samples were compared by one sample t-test for paired measurements or ANOVA and Tukey’s test for multiple categories. Non-normally distributed samples were compared using the Friedman test for multiple category repeated measurements, Kruskal-Wallis test for multiple category single measurements, or the sign test for skewed paired measurements. The Friedman and Kruskal-Wallis test were used with Nemenyi post hoc tests to determine pairwise differences between categories. t-Tests and sign tests were adjusted using Holm’s method for correcting for multiple comparisons. For statistical analyses involving comparisons of the behavior of males and females, we repeated the tests after accounting for differences in classifier accuracy by changing the proportion of behavior by this difference (i.e. 4.93% for probing and 3.25% for walking) and confirming that the results were the same. Sample sizes followed conventions in the field. For experiments with multiple stimuli presented to each animal, 4–6 days of data were collected. For endpoint and single stimulus experiments, 7–11 days of data were collected.

Logistic regression models for the blood blanket experiment were trained using the Python sklearn package with the proportion of time mosquitoes spent in each of the four behaviors (groom, walk, probe, and fly) for 2 min after the light stimulus as predictors. These periods of time were –2 to 0, –8 to –6, and –14 to –12 min relative to the heat stimulus for the 2, 8, and 14 min inter-stimulus interval experiments. The dependent variable was whether the mosquito engorged by the end of the experiment. Models used the liblinear solver, random_state of 0, and balanced weight_class. Bootstrapping was performed using 10,000 resamples with replacement of the engorgement dataset to determine the distribution of predictive models; 10,000 shuffles of the engorgement data were used to determine whether the predictive model performed above chance. Leave-one-out cross-validation was used to determine whether the model was overfitted.

All data generated or analyzed during this study are included in the manuscript and in Figure 1—source data 1, Figure 2—source data 1, Figure 3—source data 1, and Figure 4—source data 1. Large datasets are available at https://github.com/trevorsorrells/Optothermocycler, (copy archived at swh:1:rev:9c70d0c348ad6d8a32663ca23257616f915ab06e; Sorrells, 2021).

Analysis code used in this publication is available at https://github.com/trevorsorrells/Optothermocycler; Sorrells, 2021.