Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti.

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for materials, resources and reagents, including mosquito lines, should be directed to and will be fulfilled by the Lead Contact, Jeff Riffell ( jriffell@uw.edu ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS Wild type

Aedes aegypti mosquitoes (line Rockefeller F25, MR4–735) were used for the tethered flight experiments. The colony was maintained in a climatic chamber at 25±1°C, 60±10% relative humidity (RH) and under a 12–12h light-dark cycle. An artificial feeder (D.E. Lillie Glassblowers, Atlanta, Georgia; 2.5 cm internal diameter) supplied with heparinized bovine blood (Lampire Biological Laboratories, Pipersville, PA, USA) placed on the top of the cage and heated at 37°C using a water-bath circulation, allowed us to feed mosquitoes on weekdays. Cotton balls soaked with 10% sucrose were continuously provided to the mosquitoes. Groups of 200 larvae were placed in 26×35×4cm covered pans containing tap water and were fed on fish food (Hikari Tropic 382 First Bites - Petco, San Diego, CA, USA). Groups of 120 pupae were then isolated in 16 Oz containers (Mosquito Breeder Jar, Bioquip Products, Rancho Dominguez, CA, USA) until emergence. Adults were then transferred into mating cages (BioQuip Products, Rancho Dominguez, CA, USA) and maintained on 10% sucrose. Mosquitoes used in the calcium imaging experiments were from of the Ae. aegypti Liverpool strain, which was the source strain for the reference genome sequence. Briefly, this mosquito line was generated by injecting a construct that included the GCaMP6s plasmid (ID# 106868) cloned into the piggyBac plasmid pBac-3xP3-dsRed and using Ae. aegypti polyubiquitin ( PUb ) promoter fragment. Mosquito pre-blastoderm stage embryos were injected with a mixture of the GCaMP6s plasmid described above (200ng/ul) and a source of piggyBac transposase (phsp-Pbac, (200ng/ul)). Injected embryos were hatched in deoxygenated water and surviving adults were placed into cages and screened for expected fluorescent markers. Mosquitoes were backcrossed for five generations to our wild-type stock, and subsequently screened and selected for at least 20 generations to obtain a near homozygous line. The location and orientation of the insertion site was confirmed by PCR (see [ 33 ] for details). To characterize the expression of GCaMP in different cell types in the AL, we double-stained for GFP (for the GCaMP6s; Abcam, Cambridge, MA, USA – Cat. no. ab6556; 1:1000 concentration) and glutamine synthase (GS; a glial marker; Sigma-Aldrich, St. Louis, MO, USA - Cat. no. MAB302; 1:500 concentration). Double-labelling of GFP (for GCaMP6s) and glutamine synthase (for glia) revealed that ubiquitous expression of GCaMP occurred in glia, local interneurons, and projection neurons. However, glia-like processes occurred on the exterior ‘rind’ of AL glomeruli and was restricted compared to the GFP labelling, thus enabling us to record from the central interior regions of the glomerular neuropil ( Figure S3F ). Similarly, GFP was strongly expressed in the optic lobe lamina and other loci, with restricted GS-labeling and little overlap ( Figure S3F ). In both brain regions, the GCaMP6s expression was very high in lobula cell types and AL projection neurons (PNs), such that during stimulation the cells could be imaged and tentatively reconstructed via optical sectioning. For all the experiments, 6–8 day old female mosquitoes were used. For behavioural experiments, this gave mosquitoes the time to mate in the containers before the tethered flight experiments (random dissection of females revealed that 95% of them had oocytes); all experiments in the flight arena occurred during the last three hours of the mosquitoes’ subjective day [ 52 – 55 ]. Female mosquitoes used in calcium imaging experiments were unmated and kept in isolation allowing fine-scale control of their age, reproductive status, physiological state, and sugar feeding. Previous studies have shown no differences between mated and unmated females in their host-seeking responses to odour cues [ 55 ]; as a first step we wanted to ensure that any neural modulation was due to the stimuli presented to the animals. Sugar feeding (10% sucrose) up to 16 h before experiments increased the calcium fluorescence and duration of the experiments. METHOD DETAILS Tethered Flight Visual Arena Tethered flight responses by mosquitoes to olfactory and visual stimuli were tested in an LED-based arena ( sensu [ 18 ]; Fig 1A ). The arena consists of an array of 96×16 LEDs, each subtending 3.75° on the eye, subtending 360° horizontally and 54° vertically. Mosquitoes were cold anesthetized on ice and tethered to a tungsten wire using UV-activated glue (Loctite 3104 Light Cure Adhesive, Loctite, Düsseldorf, Germany) applied on the thorax. The main body axis was positioned at a 30° angle from the tether. Mosquitoes were then stored at room temperature in a closed container for an approximate 30 minute recovery period. Tethered mosquitoes were centred in a hovering position within the arena ( Figure 1A ; [ 18 ]). Mosquitoes were placed directly under an infrared (IR) diode and situated above an optical sensor coupled to a wingbeat analyser (JFI Electronics, University of Chicago; [ 18 , 56 ]). The beating wings cast a shadow onto the sensor, allowing the analyser to track the motion of both wings and measure the amplitude and frequency of each wingbeat. Measurements were sampled at 5 kHz and acquired with a National Instrument Acquisition board (BNC −2090A, National Instruments, Austin, Texas, USA). Odour delivery The mosquito was centred between an air inlet and a vacuum line aligned diagonally with one another, 30° from the vertical axis ( Fig 1A ). The air inlet was positioned 12 mm in front of and slightly above the mosquito’s head, targeting the antennae from an angle of 15°. The vacuum line was positioned behind the mosquito 25 mm away from the tip of the abdomen. Two different airlines independently controlled by a solenoid valve (The Lee Company, Essex, CT, USA, LHDA0533115H) intersected this main air inlet, one delivering nitrogen and, the other, CO 2 . Mass flow controllers for both the CO 2 and nitrogen delivery allowed for the CO 2 to be set at different concentrations (0, 1, 2.5, 5 and 10%) and pulse durations. Nonanal was diluted at 1:100 in mineral oil and 2 μL was pipetted on to a filter paper (2M Whatman) in a Pasteur pipette. Mosquitoes responses to different CO 2 concentrations and pulse durations For these experiments, a visual pattern of alternating vertical bars comprised of either inactive or fully-lit LEDs, each 16×6 pixels in size (i.e. 22.5° wide, 54° tall) was used. The pattern was briefly placed in closed-loop at the beginning of the experiment in order to encourage the mosquitoes to fly and then held motionless during the presentation of CO 2 . Closed-loop control of the pattern position was achieved using the difference between the left and right amplitude signals. Concentrations of 5% and 10% CO 2 were initially tested, delivered for durations of 20, 10, 5, 1, and 0.5 seconds. One second pulses of CO 2 at 2.5% and 1% were also tested. Potential mechanical stimulation associated with the onset of the pulses was controlled for by delivering N 2 pulses for all the tested durations. Because a 1 sec pulse of 5% CO2 was sufficient to produce a reliable, robust frequency response, this was the concentration and pulse duration used throughout the remainder of this study. Moving visual patterns To test the response to looming and drifting objects, large-field patterns of optic flow, and rotating field patterns, we adapted a broad panel of visual stimuli that are known to be important for guidance and stability during flight in other insects [ 29 ]: looming and fading squares, progressive and regressive bars, and starfield patterns (75% of pixels ON), yaw, a 22.5° wide square-like object (6×6 pixels, 20.25° tall) or a 22.5° wide and 54° tall bar moving either from left to right (Clockwise; CW) or from right to left (Counter-clockwise, CCW) ( Figures 1 and S2 ). The stimuli were each presented for two seconds and were separated by a 4 sec period during which all LEDs in the arena were lit. The angular velocity of objects moving on the display was 150°/sec. The entire experiment consisted of five trials of twelve visual stimuli presented twice (either immediately following a 1 sec pulse of CO 2 , or alone), the order of which was randomized at the beginning of each trial, using Matlab’s random number generator.

Dynamics model

To quantify the changes in visuomotor turning dynamics elicited by CO 2 , we modelled their behaviour using the approach described by Reichardt and Poggio [ 32 ]. Reichardt and Poggio describe the closed-loop behaviour of a tethered insect steering towards an object with the following dynamics: Eqn. 1 Θ ψ ¨ ( t ) + k ψ ˙ ( t ) = N ( t ) + S ( t ) − R ( ψ ( t ) , t ) , Where ѱ( t )is the angular position of the object on the mosquito’s retina, Θ is the mosquito’s moment of inertia, k is the aerodynamic friction, N ( t ) is mean-zero gaussian noise, S ( t ) describes the motion of the object relative to stationary objects in the world, and R (ѱ( t )), t ) describes the mosquito’s steering response. The steering response, R (ѱ( t )), t ), is a nonlinear function of the object’s position and velocity on the retina, which may be approximated by [ 32 ]: Eqn. 2 R ( ψ ( t ) , t ) = r ( ψ ( t ) ) ψ ˙ ( t ) + D ( ψ ( t ) ) , where r ( ψ ( t ) ) ψ ˙ ( t ) describes the mosquito’s response to the velocity of the object, and D (ѱ( t )) describes the mosquito’s response to the position of the object. The advantage of using open-loop data is that they provide information over the entire range of ѱ = [−π,π]. These data can then be used to estimate r (ѱ( t )) and D (ѱ( t )) by comparing the mosquito’s turning responses (L-R WBA) for objects moving in the clockwise (CW) and counter-clockwise (CCW) directions. The velocity component can be calculated from the difference, r (ѱ) = CW (ѱ) − CCW (ѱ) ( Figure 1F ), because the position components cancel out, whereas the position component can be calculated from the sum D (ѱ) = CW (ѱ) + CCW (ѱ) ( Figure 1G ), because the velocity components cancel out [ 37 ]. The canonical shape for r (ѱ) is a positive even function, such as a horizontal line or cosine curve, and D (ѱ) is typically an odd function, such as a line with a positive slope or sine curve (corresponding to saturation at peripheral angles) [ 32 ]. These canonical shapes correspond to steering responses that are simultaneously proportional to the objects position and velocity. In both the presence and absence of CO 2 , mosquitoes’ responses are proportional to the object’s position and velocity, corresponding to object tracking. The precise shape of D (ѱ) and the magnitude of r (ѱ), however, changes in the presence of CO 2 . To characterize these changes, we modelled r (ѱ) as a cosine, and D (ѱ) as a sine curve ( Figure S2F – G ). The cosine approximation of r (ѱ) is not perfect, however, the changes in magnitude are appropriately reflected in the model. For the square, CO 2 had little effect on r (ѱ), whereas it significantly increased the frequency of D (ѱ) (p=0.003), contracting the sinewave, which corresponds to an increase in the slope of the proportional response when ѱ is in front of the animal. For the bar, CO 2 significantly increased the magnitude of r (ѱ) (p=0.007), corresponding to an increase in the velocity dependent response, and modestly increased the frequency of D (ѱ) (p=0.06). How might these changes in open-loop responses relate to free flight behaviour? To gain a better intuition for how the functions r (ѱ) and D (ѱ) shape the mosquito’s behaviour, we simplified the dynamical system to bring it into a standard form. If we consider the mosquito interacting with a static object with some initial condition, we can eliminate N ( t ) and S ( t ) in Eqn. 1 , since both are equal to zero, leaving us with the following nonlinear second order differential equation: Eqn. 3 Θ ψ ¨ ( t ) + ( k + r ( ψ ( t ) ) ) ψ ˙ ( t ) + D ( ψ ( t ) ) = 0. We used cosine and sine approximations of r (ѱ) and D (ѱ) in Figure S3 to linearize the system about the stable equilibrium, ѱ = 0, allowing us to approximate r (ѱ) as a constant, and D (ѱ) as a line, resulting in: Eqn. 4 Θ ψ ¨ ( t ) + ( k + r 0 ) ψ ˙ ( t ) + d s ψ ( t ) = 0 , where r 0 = r (ѱ) = 0 and d s is the slope of r (ѱ) | ѱ=0 . This is a classic second order differential equation, equivalent to a mass-spring-damper system in which the slope of D (ѱ) determines the natural frequency and the magnitude of r 0 determines the damping. These parameters can be used to calculate how quickly the system responds to a step input, such as a mosquito seeing an object and steering towards or away from it. Larger values of d s will reduce the response delay and increase the amount of oscillations, and larger values of r 0 will reduce the extent of any oscillations, thereby increasing the stability. The results of our analysis suggest that CO 2 modulates flight behaviour such that mosquitoes respond to visual objects with faster and more stable responses. To illustrate this, we numerically integrated Eqn. 3 using the sine and cosine curves from Figure S2 for r (ѱ) and D (ѱ) ( Figure 1G ) to simulate a mosquito turning towards a fixed object. Because the dynamics for tethered flight are slower than free flight, and because of values for r (ѱ) and D (ѱ) are in relative units (based on the amplifier gains in the wing beat analyser), we chose values for Θ and k to emphasize the relationship between r (ѱ) and the stability. The values we chose were Θ = 1 (relative units) Θ k = 3 sec . The ratio Θ k represents the time constant of the passive rotational dynamics; values smaller than 1 ensure that the oscillations would be damped even with a small value for r (ѱ). The simulations show that CO 2 increases the speed of mosquitoes’ responses to squares, at the expense of stability, and CO 2 dampens the oscillations of mosquitoes’ responses to bars, increasing the stability at the expense of speed. Because the magnitude of the velocity response function was larger for bars ( Figure 1F ), and because this modulation increased the stability of the response, we chose to use bars as our primary visual stimulus for the calcium imaging. Although it seems as though CO 2 has opposite effects on the dynamics for the bar and bar (compare Figures 1G and S2E ), there are several explanations for this. First, the mosquitoes’ behaviour in response to the square was more variable, and the changes more subtle, thus the response may not be representative of their free behaviour. Second, it is possible for one dynamical system to have opposite effects with an increase in gain depending on the initial gain. For example, when the system G ( S ) = (s +10)(s + 0.5 ± 1.5 j )/((s + 0.001)(s + 0.5 ± 0.5 j )) transitions from low to intermediate gain the stability decreases, but when it transitions from intermediate to high gain, the stability increases. If the mosquito’s responses to bars and squares resulted in high and low initial gains, respectively, and the gain for each response increased multiplicatively due to CO 2 , it would explain our results. However, we do not have sufficient data to test this hypothesis at present.

Calcium imaging

Image acquisition: Visual and odor-evoked responses were imaged in the lobula region of the mosquito optic lobe, and the antennal lobe region, taking advantage of our genetically-encoded ubiquitin-GCaMPs mosquito line [ 33 ]( Figures 2A – C ; 3H ). Calcium-evoked responses were imaged using the Prairie Ultima IV multiphoton microscope (Prairie Technologies) and Ti-Sapphire laser (Chameleon Ultra; Coherent). The laser power was adjusted to 20mW at the rear aperture of the objective lens (Nikon NIR Apo, 40X water immersion lens, 0.8 NA), and bandpass filtered the GCaMP fluorescence with a HQ 525/50 m-2p emission filter (Chroma Technologies) and collected the photons using a multialkali photomultiplier tube. Images were collected at 2 Hz for each visual and visual+odour stimulus, for a total duration of 350 s ( Figure 2 ), and calcium-evoked responses are calculated as the change in fluorescence and time-stamped and synced with the stimuli. Individual mosquitoes were tethered to a holder, and their cuticle removed to provide access to the antennal lobe or lobula regions of the brain [ 56 ]. The mosquitoes were placed at the centre of a semi-cylindrical visual arena (frosted mylar, 20 cm diameter, 20 cm high); a video projector (Acer K132 WXGA DLP LED Projector, 600 Lumens) positioned in front of the arena projected the visual stimuli. To separate the wavelength of the light emitted by the projector from the GCaMP6 fluorescence, we used the projector’s blue channel (peak at 451 nm, 18 lux, 0.02 W/m 2 ) and further reduced the longer wavelength component by covering the projector with three layers of blue gel filter (ROSCOLUX #59 Indigo). Select visual stimuli were the same as those used in the arena experiments: a bar, square (15°) and star-field pattern (comprising 75% of the screen). Image analysis for Lobula ROIs: The ubiquitous expression of GCaMP6s made it difficult to distinguish between different cell types in the imaging planes. We thus used a series of criteria and image analyses to select ROIs manually. To ensure that mosquitoes were viable, we used animals that showed both odour-evoked changes glomerular fluorescence in the AL and changes in lobula fluorescence from stimulation with strong puffs of air to the head (via hand-held syringe) and from presentations of visual stimuli. Images were initially examined in ImageJ and imported into Matlab for alignment using a single frame as the reference at a given imaging depth and subsequently registered to every frame to within ¼ pixel, and subsequently Gaussian filtered (2×2 pixel; σ = 1.5–3). For detection of the calcium dynamics, pixels were chosen based on fluorescence changes above the background threshold (1.02 to 15.9-times the baseline fluorescence), and ROIs were manually selected based on pixel intensities and appearances similar to axonal regions; restricted ROI surface areas (40–100 μm 2 ) were used to minimize recording multiple cells. Images were acquired at approximately 40 to 100 μm from the ventral surface ( Figures 2 and 3 ) – neuropil in this region showed strong responses to visual stimuli, and odour-evoked modulation –, and optical sections (1 m) were taken to tentatively reconstruct axonal regions associated with the regions of interest (Amira v.5, Thermo Fisher Scientific). We typically had stable imaging for approximately 1.5 h allowing complete testing of the experimental series. Image analysis for AL ROIs: Antennal lobe ROIs were selected mainly based on the criteria listed above, except ROI selection was based on the clear delineation between glomerular boundaries. Glomerular ROIs were imaged at 40 μm from the ventral surface. Glomeruli at this depth show strong responses to either host- or plant-related odorants. For instance, the lateral cluster of glomeruli (AL3, LC2, V1) are especially responsive to host odorants, including nonanal, octanal, and hexanoic acid, and to a lesser extent, CO 2 (AL3, a glomerulus that is broadly responsive to stimuli), whereas glomeruli in the anteriomedial cluster respond to plant-related compounds, such as linalool, lilac aldehyde and myrtenol. At this depth, 14–18 glomeruli were neuroanatomically identified and registered between preparations. Calcium-evoked responses are calculated as the change in fluorescence and time-stamped and synced with the stimulus pulses. After an experiment, the AL was serially scanned at 1 μm depths from the ventral to the dorsal surface to provide glomerular registration to our tentative AL atlas (n = 6 female mosquitoes) as well as one that was previously published [ 57 ]. We note that glomeruli identified in our imaging experiments did not always conform, regarding glomerular number and position, to the previously published atlas; however, the two atlases provide a first principles approach for identifying and registering glomeruli.

QUANTIFICATION AND STATISTICAL ANALYSIS

Analyses were performed in R. For each stimulus, a baseline wingbeat frequency was determined by averaging the frequency across a 1 sec time window preceding the stimulus delivery (either visual or olfactory, according to the experiment) and then subtracting this value from the max frequency values following the stimulus. Trials were discarded in which the mosquitoes stopped flying, indicated by a drop in wingbeat frequency below 200 Hz. The mean response for each individual was calculated from the saved trials and used as a replicate to calculate the mean response for each treatment group. This latter was calculated using the difference in frequency, turning tendency (L-R WBA), and total amplitude (L+R WBA) before and after the stimulus. One-tailed Student’s t-tests for paired samples were used to test for differences from baseline and t-tests for independent samples were used to test for differences between groups. As stimuli that were presented with two directions of movement (i.e. square, bar and yaw from left to right or from right to left) did not elicit significantly different responses (Student t test; 0.061.15; 40