In his Correspondence article, Dr Packard questions our approach to studying cuttlefish camouflage body patterning (Chiao et al., 2010). While we respect Packard's invaluable contribution to the study of cephalopod chromatophores and freely acknowledge that he has inspired us to continue to investigate them, it seems to us that his criticism of our paper misses the point. His major concern was that cuttlefish did not match the brightness of the artificial substrates in most of our experiments, but our experiments were specifically concerned with pattern and contrast, not brightness. The reason for using these carefully designed substrates was not to test all aspects of ‘background matching’ but rather to apply psychophysical methods to examine systematically the visual features of the background that elicit the pattern design in Mottle camouflage, a particular type of body pattern whose function falls within the scope of general resemblance to the background [or what Stevens and Merilaita term ‘background matching’ (Stevens and Merilaita, 2009)].
Earlier experiments using checkerboards to study the dynamics of body patterns in flounders and cuttlefish have demonstrated the clear advantages of using artificial backgrounds to investigate the visual sampling rules used by camouflaging animals (Chiao and Hanlon, 2001; Ramachandran et al., 1996) and this was the starting point for our investigation. In the artificial substrate experiments in our paper, quantification of substrate and body patterning was carried out to categorize the effect of specific visual cues on pattern expression (which is only one aspect of the overall ‘body pattern’ that is defined as the overall appearance of the animal, to include pattern, color, brightness, contrast and skin texture).
It is crucial to realize that the artificial substrates used in our experiments lie outside the luminance space of substrates that cuttlefish have evolved to match; i.e. none of the substrates that cuttlefish encounter in their natural habitats are simultaneously as high in average reflectance and in contrast as the substrates on which they were tested in this study. There is thus no reason to suppose that the animals will match both the textural properties and the brightness of these checkerboard substrates. To achieve even a rough brightness match to these artificial substrates, our animals would have had to deploy very light uniform patterns that would have failed to match any of the textural properties of the substrates. Instead, as we have documented, they showed a very strong tendency to match the granular structures rather than the brightness of the substrates. That they preferred to make these pattern matches at the expense of brightness matches testifies to the potency of pattern in controlling their responses.
We made reference to this issue in our explanation of supplementary material figure S1 (Chiao et al., 2010), where we compared the granularity of each artificial substrate with the granularity of the animal's body pattern. We stated on p. 197 that, ‘It is apparent that the overall shape of the granularity spectrum of the backgrounds is similar to that of the animals. However, close examination of these curves reveals that the magnitude and the peak of the backgrounds do not exactly match that of the animals, even in the case of natural substrates.’ Thus we did not ‘apparently miss’ that the animals are not exactly matching the background as Packard asserts. Moreover, we have published numerous images from the laboratory and the field showing the brightness match of various cephalopods to many backgrounds (e.g. Hanlon and Messenger, 1988; Hanlon and Messenger, 1996; Hanlon et al., 2009).
Additionally, our granularity statistic method was not designed to capture the overall reflectance of body patterns, because the mean intensity is subtracted out before analyzing the image of the animal in different spatial frequency bands. This method addresses a different problem: it provides a measure of the size and contrast of the light and dark patches in the skin [see Barbosa et al. (Barbosa et al., 2008) where the method was introduced] (see also Spottiswoode and Stevens, 2010). Although this particular quantitative approach to define statistically the main pattern types deployed by cuttlefish ignores the ‘tone matching’ between animals and backgrounds in this paper, our previous research has emphasized that mean substrate intensity (among other factors) plays an important role in modulating cuttlefish body patterns (Chiao et al., 2007).
Packard's concern with the distinction between morphological and functional approaches is understandable, although he may have misunderstood our efforts entirely. We recognized his neurophysiological work in determining the skin patch organization of chromatophores as the ‘physiological units’ (Packard, 1982), and his explanation of the production of Mottle body patterns by lateral inhibition and neuromuscular gain controls (Packard, 1995). However, the important facet of our analysis of the Mottle body pattern for this paper lies in the animal's ability to control both small- and large-scale Mottle skin components to resemble the size scale and contrast of light and dark objects in the immediate visual background. Although Packard's observations, such as exposing cuttlefish to CO2-bubbled seawater in his supplementary Movie 1, could distinguish whether ‘screens and mottles’ are functionally different, we consider cuttlefish to be camouflaged only if the animals show stable body patterns while stationary on both natural and artificial substrates. We believe these criteria enable us to reveal key background visual features that cuttlefish detect and respond to for camouflage, and not to secondary defenses such as deimatic or protean behaviors that were evoked in his Movie.
While it is true that our use of 1000 lx illumination may exceed the amount of light that cuttlefish would encounter in most natural habitats, our recent research indicates that their camouflage body patterns on the various artificial substrates remain the same as reported here when subjected to lower light levels, all the way to starlight, 0.003 lx (J. J. Allen, L. M. Mäthger, K. C. Buresch, T. Fetchko, M. Gardner and R. T. Hanlon, submitted). As for Packard's conclusion that our experiments ‘are too static’, we contend that effective camouflage patterns are indeed static when the animals are settled on a background with unchanging light fields. We are keenly aware of the dynamic nature of cephalopod adaptive coloration (Hanlon, 2007) and have been building a library of high-definition video of cuttlefish and octopus as they forage in highly diverse natural habitats worldwide. Such footage – representing hundreds of hours of observations under natural lighting fluctuations – has guided our laboratory experimentation from the outset. In this respect, our approach to studying cephalopod adaptive camouflage complements that of Packard.
In conclusion, our goal in this paper and other recent publications has been to study experimentally the visual cues that might elicit certain patterns in cuttlefish. When doing experiments there is inevitably a tradeoff between reducing the number of variables and obtaining a biologically meaningful result. It is a pity that Packard does not seem to recognize this dilemma in his critique of our work.
