Ontogeny of the carp (Cyprinus carpio L.) innate immune system: Gene expression and experimental limitations

The objective of this study was to investigate the ontogeny of the immune system in common carp (Cyprinus carpio, L.). The work has been focused on innate immune responses during the wound healing processes and how the innate immune response develops with age and size of the fish.
Newly hatched carp were brought to the facilities at DTU and kept in aquaria at 25°C. They were initially fed Artemia nauplii, and later switched to commercial dry granulate feed. Carp were anaesthetised and then experimentally wounded at days 10, 16, 24, 47 and 94 days post-hatch. Sampling was carried out at day 1, 3 and 7 post-wounding and samples were stored in RNA later for isolation of RNA. The physical tissue damage was performed using a sterile needle, which penetrated the skin and the underlying musculature in an area above the lateral line of the left side of fish. Carps at the age of 10, 16 and 24 days post-hatch were stored and processed whole, whereas just the muscle (the left (wound area) and right filet (internal control)) was sampled for the two latter time-points. mRNA was extracted from the samples, cDNA was synthesised and gene expression was quantified using real-time RT-PCR. The investigated genes were IL-1β, IL-6, TNF-α, SAA, Hsp70, TGF-β and the mucins Muc2c and Muc5bc.
It can be generally concluded that the response of the investigated genes appeared to be faster and more pronounced at earlier life stages. However, care should be taken when comparing the results for the fish wounded on days 10, 16 and 24 post-hatching with the fish wounded on days 47 and 94 post-hatching since the way of sampling differed. However, it is inherently difficult to standardise experimental procedures for fish that increase 1000-fold during the course of the experiment. The results and experimental pitfalls will be further discussed.