Over the course of the day, we exchanged the behavior arena seawater several times with water sourced from the Marine Resources Center (MRC) recirculating seawater system. Temperature varied by ~5°C maximum. We did not measure ammonia and pH throughout. Water quality changes were likely to be slow, and we noticed no trend with increased time out of the recirculating water system (hunting improved over time as the animals acclimated to the tank). Animals were motivated with live shrimp rewards and ate up to 30 grass shrimp (1.5 to 3 cm in length) per day.

We developed a bioassay to train cuttlefish to hunt the image of shrimp presented on a screen. The setup ( Fig. 1A ) consisted of a computer monitor (Dell Ultrasharp LED U2413 24″ Premier Color) positioned against the side of a plastic tank (IRIS USA File Box, model no. 586490, 10.75 inches deep × 13.88 inches wide × 18.25 inches long) used as the experimental arena. Two cameras monitored the behavior of the animal. A high-speed camera (Photron SA3 or Photron FASTCAM Mini WX100 with Canon EF 24 to 70 mm f/2.8L USM macro lens) positioned over the tank captured the entirety of the arena at 250 or 500 frames/s. In addition, an underwater camera (GoPro Hero5 or Hero7 with Super Suit) placed inside the tank on the side opposite to the monitor provided an additional vantage point (see movie S1). After establishing the camera location and lens focus each day, we took an image of a ruler at the bottom of the tank before the experiments started. This served as the scale bar that allowed us to measure distances in x-y dimensions in the tank.

We withheld food for 2 days before training, beginning to motivate hunting and expedite learning. During the initial training stages, a live grass shrimp (Palaemonetes vulgaris) reward was delivered to the cuttlefish for each attempt by the animal to engage with the image of a moving shrimp presented on the screen (black shrimp on a white background). In the subsequent training stages, shrimp rewards were restricted to trials during which the cuttlefish responded to the on-screen target by extending its tentacles, i.e., it entered hunting mode and was preparing to strike at a target deemed suitable to capture. Once this behavior became consistent, we affixed a Velcro patch (approximately 1 cm 2 ) to the dorsal surface of the animal’s head. We achieved this by netting the animals out of the tank, patting the skin dry with a paper towel three times, and applying a superglue-covered Velcro patch directly to the skin and holding in place for 10 s. Immediately after returning the animals to the tank with care, we fed them with a large grass shrimp. Subsequently, we repeated training as detailed above, except that we only gave a shrimp reward when the animal struck out its tentacles attempting to catch the on-screen prey. Once given, behavior was consistent, a custom-made pair of glasses (see below) was placed onto the animal, attached via the Velcro patch, and training was repeated. A few hours after being fitted with glasses, some animals would reliably hunt, while other more cautious animals or those initially not interested in viewing the screen took up to 2 days to reliably interact with shrimp video stimuli. We tested trained animals with positive and negative disparate stimuli as soon as five successful strikes of the black shrimp on white background video were completed. We repeated trials over the course of several days.

Visual stimuli

We constructed anaglyph glasses for the cuttlefish, adapted from those described by Nityananda et al. (6) for mantids (Fig. 1B, right eye: red = Lee 135 Deep Golden Amber; left eye: blue = Lee 797 Purple; see movies S1 and S2). Each filter allowed transmission of a subset of the spectrum of light while blocking or reducing other wavelengths. Note that we used a double layer of red and blue filters, instead of a single layer, to better separate the wavelengths reaching each eye. A Velcro patch on the underside of the glasses allowed easy attachment or removal of the glasses to the animals. This paradigm allowed us to selectively stimulate both the left and right eyes and create an offset between the right and left images to produce an illusory depth percept for animals that use stereopsis (Fig. 1C). However, we later found the amber filter to be at the far end of the cuttlefish opsin sensitivity; therefore, luminance was not matched between the two eyes. This, however, did not appear to affect the cuttlefish’s ability to detect a stereoscopic stimulus. This concurs with a previous report that the shape of the prey is more strongly discriminated by cuttlefish than the brightness of the prey (30). To remediate the intensity difference, in the second batch of animals/experiments, we used blue and green filters (right eye: green = Lee 736 Twickenham Green; left eye: blue = Lee 071 Tokyo Blue; as a double layer). We chose to use these blue and green filters as they had better intensity matches and more similar photon catches for cuttlefish, i.e., lower the chance that intensity differences between the two eyes would provide cues or create distraction artifacts. We repeated the behavioral tests with the blue/green filters. Five of the six animals that engaged with the random-dot stimuli experiments wore the blue and green filter combination. The behavioral results were the same between the animals wearing the two types of glasses, so we pooled the datasets together.

We referred to a positive disparity as disparity between images that causes the illusory depth percept to appear anterior to the screen, whereas a negative disparity will create a percept behind the screen. The value attributed to a disparity stimulus (1, 2, or 3 cm) indicates the offset between the images presented to each eye. For each animal, we tested a range of stimulus disparities and experimental protocols in a random order over the course of each experimental day.

We created the shrimp stimulus presented to the cuttlefish via the screen from videos of grass shrimp recorded underwater using a GoPro camera. We then converted this video into a gray scale. By duplicating some of the video frames but shifting the shrimp image location along the x axis, we created a clip of a shrimp traveling the full width of the screen. Then, we duplicated and flipped along the x axis all the frames of the video. When played consecutively, this made the shrimp “flip direction” and return walking toward the starting point (left side of the tank). We used this forward and return walk across the screen by the shrimp as the basis of most stimuli. To generate the silhouette of a black shrimp against a uniform white background, we converted the forward and return video into a binary format. The three LED channels of the screen (fig. S1B) were used individually or as a combination of two LEDs to create six possible colors of shrimp stimulus presented against a white or a black background (fig. S1C). Radiance spectra were measured using a National Institute of Standards and Technology calibrated Avantes AvaSpec 2048 Single-Channel spectrometer coupled to an Avantes ultraviolet-visible 600-μm fiber (numerical aperture = 0.22, acceptance angle = 25.4, and solid angle = 0.1521) positioned at a distance of 125 mm from the tank wall and monitor. We collected spectra by averaging 100 repetitions of 50-ms light integration time and smoothed using an eight-point moving average filter. We made the measurements in air, rather than in water, for equipment preservation purposes. The spectra of all stimuli were measured from a full screen of color matching RGB values for individual components of all stimuli videos (fig. S1C). Measurements were repeated with the addition of either blue or red filters by positioning the glasses in the light pathway from the screen to the fiber, a few millimeters from the fiber end (fig. S2, A and B). We also repeated measurements for the blue and green filters.

The spectral sensitivity of the S. officinalis visual pigment was calculated using the equations formulated by Stavenga et al. (31) using a peak wavelength λ max = 492 nm for the α wave of the template and a peak wavelength λ max = 360 nm for the β wave (29). The relative photon catch for each color of shrimp stimulus, that is, the number of photons (N) absorbed by a cuttlefish photoreceptor for a given stimulus, was obtained using the following equation (32) N = ∫ ( 1 − exp ( − kS ( λ ) l ) ) × R ( λ ) d ( λ ) where k is the quantum efficiency of transduction = 0.0067/μm (33), S(λ) is the spectral sensitivity of the visual pigment, l is the length of the rhabdom = 400 μm (34), and R(λ) is the measured radiance spectra of the stimulus on the screen. As our goal was to produce stimuli that would only be detected by either the left or the right eye of the cuttlefish when wearing the glasses. We assessed the cross-talk of these stimuli by establishing the ratio of the quantum catch by the eye supposedly blind to the stimulus and the quantum catch of the eye intended for the stimulus (figs. S2, A and B, and S3, bottom row). Our calculations show cross-talk between stimuli to be between 1 and 24%. Note the considerably lower cross-talk for the green/blue glasses (fig. S3; see movie S4). It is important to note here that other species perceived the 3D effect with anaglyph color glasses, although they suffered photon leak of the channels (i.e., cross-talk), which can be quite large when used for humans with color vision (6, 35). Thus, detection of light via the “blocked” channel did not preclude the stereopsis test from being valid.

For experiments in which we presented a video of a shrimp against a uniform background, we tested a dark silhouette against a white background as well as a light silhouette against a dark background (figs. S2, A and B, and S3). The 4-cm-wide shrimp subtends 17.74° when viewed at 12.5 cm, corresponding to the average distance of the eyes to the screen at the start of the ballistic strike. In the case of a white background, the band of light reflected from a green shrimp was not transmitted through either the blue or red filter, resulting in a contrast between the absence of light of the shrimp appearing dark against a light background. If the shrimp is presented as cyan, then the red filter will make the shrimp appear dark against a light background for the right eye, while the blue filter will transmit the light, thus removing the contrast between the shrimp object and background, thereby rendering the shrimp indistinguishable for the left eye. This same effect is reversed for the blue and red filters with a yellow shrimp. In the case of a dark background, a magenta shrimp will transmit light to both eyes, thus contrasting against the absence of light from the surrounding black. Blue and red lights, however, will only be transmitted through the blue and red filters, respectively. We also varied other parameters of the visual scene: The shrimp moving across the visual scene either walked or swam. We tested walking and swimming shrimp to demonstrate that the hunting behavior was not constrained to a particular stimuli video or movement (fig. S4A). We tested positive and negative contrasts to demonstrate that the motion detection system was not constrained (fig. S4B). We extracted other features of the behavior (fig. S5, A to C) to tease apart how the animal behaved and reacted to stimuli. To test for the mechanism subservient to the stereo ability, we followed and adapted the methodology in Nityananda et al. (25). Briefly, we used the walking shrimp video but covered the shrimp silhouette with a random black-white dot pattern and “camouflaged” it against the background with the same type of pattern. The dot pattern shown to the two eyes was either correlated (same contrast polarity), anticorrelated (flipped contrast for one of the two eyes), or uncorrelated (different patterns for each eye). The 1.4-mm dot stimuli, when viewed at 18 cm, subtends 0.44°. The 5-mm dot stimuli, when viewed at 18 cm, subtends 1.59°. Data from both size dot patterns were combined in Fig. 3, as we found no significant difference between these datasets (correlated 0- and 2-cm disparity, P = 1.0 and 0.1074; anticorrelated 0- and 2-cm disparity, P = 0.9986 and 0.5313; see movie S4). For these dotty shrimp stimuli and backgrounds, one cuttlefish wore the red/blue glasses, while five animals wore the green/blue glasses described above. We did not find a significant difference between animal positioning locations for the 0- and 2-cm disparity conditions for the red/blue and green/blue glasses (fig. S8).

We found that 5 of 11 animals did not engage in hunting when presented with the dotty task, even when the stimulus was correlated and had zero disparity. We interpreted that the difficulty of this test, imposed by similarities between the prey and background patterns, rendered those individuals unwilling to attempt it even if they had the ability to do so, as shown by the other six animals. The responses were animal specific and consistent across days so we can exclude the hunger state. Perhaps, differences in individual character, known to exist in cephalopods, may drive the differences in motivation (36–38).