Experiment 1

The subjects of this experiment were five laboratory-reared adult/senescent Sepia officinalis (15–19 months post-hatching), housed in a round tank (823 L) containing a variety of plastic plants. For the experiment, individual cuttlefish were transferred from group housing in a large (approximately 10–12 foot diameter pool) to a rectangular experimental tank (240 L). A sleeping site was prepared using a submerged clear plexiglass “V,” with the bottom point of the “V” running parallel with the length of the tank. Below the plexiglass “V” and within the narrow base of the “V” was a layer of crushed oyster shells. At each end of the “V,” two large stones were placed to create a single central resting site. Behind the tank, a few plastic plants and a large piece of black plastic served as a backdrop. A high-resolution, black and white camera (Sanyo VCB-3384, 30 frames/sec) was positioned directly above the sleeping site, and focused at a plane that maximally captured chromatophore activity on the dorsal surface of each animal. A second camera was placed facing the side of the tank.

At 8:30 AM on day 1 of the experiment, one cuttlefish was placed into the experimental tank for 2 days of acclimation. A short period of acclimation was used because these animals were very close to the end of their lifespan (most died within a few months after we began our experiments). It was fed 1 live fiddler crab (Uca spp); immediately thereafter, a black plastic tarp was wrapped around the entire experimental area to minimize human interaction. On the morning of day 3, the two cameras were turned on to run continuously for 48 hours. Frame data were captured on a PC with a GV-2004 video card and analyzed using accompanying software. During each minute of recording, the following behaviors were scored: ‘inactive’ (“0” buried in or lying motionless on the gravel bed) or active (“1” hovering or swimming in the water column); eyes: closed (0) or open (1); and fin movement: still (0) or moving (1). However, as the inactive/active scoring was as informative as any other single measure or combination of measures, it was used as our principle measure of rest and activity. When sleep-like states with chromatophore activation (CA; hereafter referred to as ‘sleep-like states+CA’) were observed, changes in individual chromatophore pattern components as defined by Hanlon et al [20] were tabulated second-by-second as either present or not present.

All adult cuttlefish exhibited periods of quiescence where they rested against, or buried themselves in substrate on the bottom of the aquarium (Figure 1, Table 1). If provided enough substrate, the animals would completely bury themselves, leaving only their eyes above the gravel. This state was rapidly reversible as the animals quickly moved from the bottom and began swimming when disturbed. There was considerable variability in the amounts of rest across animals and on average there were no day-night differences in activity (Table 1). For three of the five cuttlefish, a state of complete quiescence was followed by a second, sleep-like state that contained phasic motor activity, as reported previously by Duntley et al., [13], [14], [15]. The eyes appeared to rapidly move beneath closed lids, chromatophore activity (CA) suddenly intensified, and the tips of the arms curled and twitched in a way not observed when the cuttlefish was awake (Figure 2 and Video S1,S2). This sleep-like state+CA lasted on average 135 seconds (st.dev. ±23 seconds) and only occurred once (in the night, when there was minimal illumination and disturbance) for each animal during the recording period.

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larger image TIFF original image Download: Figure 1. States of arousal and quiescence in the cuttlefish Sepia officinalis. Cuttlefish exhibit clear periods of activity where the animals actively swim (A) or hover (B) and periods of quiescence (C) where they lie on the surface or are partly buried in gravel bedding with closed eyes. An adult/senescent animal is shown. https://doi.org/10.1371/journal.pone.0038125.g001

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larger image TIFF original image Download: Figure 2. A putative sleep-like state with chromatophore activity (CA) in the cuttlefish Sepia officinalis. Video still frames from a representative adult/senescent cuttlefish exhibiting changing chromatophore activity during quiescence. These 4 frames show 10 second, sequential frames beginning with quiescence (0:00) and 30 seconds of the sleep-like state+CA. The camera was positioned vertically over the animal, resting on the gravel bed; the front of the animal is pointed towards the upper right corner. Changes in chromatophore components include a darkening of the body and a disappearance of a white eye-bar (0:00–0:10), a lightening of the body, the appearance of a white square (0:10–0:20) and a subsequent disappearance of the white square (0:20–0:30). Body color components as defined as described by Hanlon et al [20]. See supporting information (Videos S1,S2) for representative videos of this sleep-like state+CA. https://doi.org/10.1371/journal.pone.0038125.g002

The overall body patterning could best be described as mottled [20], which camouflage the animals well against mixed gravel substrates; during the sleep-like state+CA, particular components of the mottled body pattern turned on and off in an irregular sequence. These body pattern changes did not appear to be random firings of uncontrolled and uncoordinated neurons. We used two strategies to quantitatively evaluate this possibility.

First, we used a dendrogram analyses to determine if the chromatophore patterns observed during this state were randomly generated. Dendrograms were created using correlation coefficient distance (Ward Linkage) showing the relatedness of chromatophore pattern components (as described in [20]) during the sleep-like state+CA(Figure 3). For example, the appearances of the white square, white head bar, and white arm triangle components were positively correlated in all three cuttlefish. These components also clustered in a previous analysis of the structure of cuttlefish body patterning [21]. Similarly, white neck spots, anterior head bar, anterior paired mantle spots, and posterior paired mantle spots usually occurred concurrently with one another in all three cuttlefish. In two of the three cuttlefish (C-Check and Tiny, Fig. 3a,b), the dark arms component occurred independently and was not correlated with any other component. With the third cuttlefish (KY, Fig. 3c), the dark arms component was associated with the presence of anterior paired mantle spots.

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larger image TIFF original image Download: Figure 3. Dendrograms showing correlation coefficient distance (Ward Linkage) between body patterning components during the putative sleep-like state+CA in three adult/senescent cuttlefish (A: “C-check”; B: “Tiny”; C: “KY”). Dendrogram similarity scores range from 100% to −100% and are equivalent to Pearson or Spearman R values of “1” or “−1”. https://doi.org/10.1371/journal.pone.0038125.g003

Second, we used an autoregressive model to determine if body pattern components, taken as a whole, turned on and off at random. For each second during the sleep-like state+CA period, we determined the total number of components that were turned on as a measure of total activity. This total activity time series data was analyzed using a Box-Jenkins ARIMA model. Each of cuttlefish had a separate time series of total activity. If the body patterning components were randomly turning on and off there would be no significant autoregressive (AR) or moving average (MA) effects. Two of the three cuttlefish showed strong AR(1) time series (C-Check and KY: p<.01 for each series; p<0.10 for the third cuttlefish, Tiny) while all three showed strong MA(1) series (p<0.05 all). The moving average (MA 1) model for all three cuttlefish had a P value of less than 0.05 (C-Check: 0.00; KY: 0.001; Tiny: 0.022). Taken together, these two analyses indicate that body patterning changes were probably not manifestations of random firings of uncontrolled and uncoordinated neurons.