Uncooked and cooked individual Penaeus monodon exposed to black or white substrates. Representatives of each treatment group, cooked and uncooked, showed a distinct colour difference that translated into a strong difference in Salmofan score. Note: relative positions of each individual in photos do not match.

Colour differentiation of uncooked P. monodon. (A) Average values of L, a and b for different treatment groups. Lightness, represented by L, has a scale of 0 (pure black) to 100 (pure white). The two components a and b determine the hue and distinguish opposing colours, where positive a is red, negative a is green, positive b is yellow and negative b is blue. The amount of these hues is known as chroma (or saturation). Treatment groups: black bars, P. monodon on black background; white bars, P. monodon on white background. (B–D) Two-dimensional plots of individual colour data from uncooked animals. L was the best differentiator of uncooked colour and was the only component that could predict the background colour. Treatment groups: filled circles, P. monodon on black background; open circles, P. monodon on white background.

Effect of exposure to black or white substrates on cooked colour of P. monodon. (A) Two-dimensional plot of the average movement within the a versus b colour space for each group during cooking. As a result of cooking, each group shifted into strongly positive a and b values, indicating the presence of red and yellow hues. The direction of colour change of the prawn exposed to black or white substrates was the same. (B) The distance between two points in three-dimensional space (ΔE) showed that the colour change of the prawns exposed to white substrates did not approach that of the prawns exposed to black substrates, indicating a strong difference in the intensity of the cooked colour.

Carotenoid quantification from hypodermal tissue from P. monodon exposed to black or white substrates. Relative quantification and percent distribution of free astaxanthin and astaxanthin esters for (A) whole hypodermal tissue, (B) insoluble pellet from hypodermal tissue and (C) the soluble protein fraction from hypodermal tissue. Total carotenoids extracted from whole tissue was similar in prawns exposed to either black or white substrates, but there was a greater proportion of astaxanthin esters in prawns exposed to white substrates. Although this effect was not significant, this trend was the same as significant effects observed in previous studies (Tume et al., 2009). The insoluble fraction contained almost exclusively astaxanthin esters, and there was no quantitative difference between substrates in any carotenoid group. Meanwhile, the soluble protein fraction contained greater than 80% free astaxanthin, and there was a significant difference in the relative quantity of free astaxanthin between prawns exposed to black or white substrates. *P<0.02; **P<0.002.

Quantification of soluble protein extracts from P. monodon hypodermal tissue exposed to black or white substrates. Equal amounts of soluble protein extract from six prawns exposed to either black or white substrates were separated on the basis of size on a 4–16% polyacrylamide gel. Band intensity was quantified for four bands of different sizes as shown. The total abundance of one band of approximately 21 kDa in size was significantly different between the treatments. This band was the same size as the CRCN protein from other crustaceans, and also cross-reacted with a specific anti-CRCN antibody (data not shown).