Regulation and Optimisation of Prawn Colouration: There is more to it than meets the eye.

1. Regulation and Optimisation of Prawn Colouration: There is more to it than meets the eye. Food Futures Flagship Dr Nick Wade

2. Effectors of Crustacean Colouration Morphological Physiological Feed additives (Astaxanthin) Environment (background colour) ?? Gene expression and localisation DARK TANKS LIGHT TANKS

3. Experimental Setup Substrate Dietary Astaxanthin BLACK WHITE 70 g/kg 70 g/kg Duration 42 days 42 days

4. Experimental Results of P. monodon Exposed to Black or White Substrates Prawn Colour Chart 10-11 4-5

5. Quantify the Colour Difference BLACK WHITE The colour shift is 6-7 grade scores!!

6. Experimental Results Substrate Dietary Astaxanthin BLACK WHITE 70 g/kg 70 g/kg Duration 42 days 42 days Animal Chromatophores Colour Grade Score 4 - 5 10 - 11

7. Change in Epithelial Carotenoids Proportion of Total (%) • Dark substrates promote the accumulation of free astaxanthin in the hypodermal tissue • Light substrates increase the proportion of astaxanthin esters.

8. Experimental Results Substrate Dietary Astaxanthin BLACK WHITE 70 g/kg 70 g/kg Duration 42 days 42 days Animal Chromatophores Colour Grade Score 4 - 5 10 - 11 Total carotenoid free Astaxanthin Astaxanthin ester EQUAL LOW HIGH EQUAL HIGH LOW

9. Colour Protein Accumulates in Animals Held on Black Substrates colour protein • Strong in abundance of the colour protein. • Colour protein, bound to free astaxanthin, was accumulated in the hypodermal tissue from • P. monodon after exposure to dark substrates.

10. No Change in Colour Gene Expression • There was no significant CRCN gene expression change across black or white treatments in P. monodon. • The ratio of the CRCN-A and CRCN-C subunits remained stable. • The expression of CRCN in P. monodon is unaffected by background substrate colour.

11. Effectors of Crustacean Colouration Substrate Dietary Astaxanthin BLACK WHITE 70 g/kg 70 g/kg Duration 42 days 42 days Animal Chromatophores Colour Grade Score 4 - 5 10 - 11 Total carotenoid free Astaxanthin Astaxanthin ester EQUAL LOW HIGH EQUAL HIGH LOW Colour protein abundance Colour gene expression RAISED LOWERED STABLE STABLE

12. What Have We Learned So Far? •  Prawns held on black substrates have dilated chromatophores, and greater levels of free astaxanthin that is associated with higher CRCN protein abundance. •  Prawns held on white substrates have constricted chromatophores, greatly elevated levels of astaxanthin esters and lower CRCN protein abundance. •  There is no change in expression of the two CRCN genes caused by exposure to dark or white substrates, or across the prawn moult cycle. •  Major differences in cooked colour were observed between animals exposed to white or black substrates, from 24 to 31 on the Salmofan chart. Elevated levels of CRCN protein in the hypodermis are essential to achieve the highest Salmofan colour scores and maximise prawn value.

13. colour protein Effectors of Crustacean Colouration Morphological Physiological Feed additives (Astaxanthin) ✔ Environment (background colour) Colour protein abundance DARK TANKS LIGHT TANKS

14. Ongoing Research • DSM • Where does the protein go and how quickly does it accumulate? • Can we further optimise dietary carotenoid levels when combined with short-term substrate colour treatment? • Seafood CRC • Does protein abundance underlie on-farm colour variability? • Test on-farm methods to maximise protein abundance and hence colour in cooked animals.

15. Acknowledgements Mike Anderson Joanne Mountford Ron Tume Simon Tabrett Nick Polymeris MelonySellars Nigel Preston Brett Glencross Jacques Gabaudan Animals kindly provided by: