Spatial and temporal distribution of the ecdysteroid receptor (EcR) in haemocytes and epidermal cells during wound healing in the crayfish, Procambarus clarkii

Wound healing in crustaceans preserves the integrity of the integument and prevents entry of pathogens. We studied the interaction between the moulting hormones (ecdysteroids) and the cellular events under the wound during wound healing with or without bacteria infection. Wounding of the carapace by abrasion induced a rapid increase in circulating ecdysteroid levels to a low sustained plateau level for about 12 days, followed by a sharp premoult peak and moulting. Within 48 h of wounding, the nuclear receptor for ecdysteroids (EcR) appeared in the nuclei of haemocytes (hyaline, semigranular and granulocytes), visualized by confocal laser scanning microscopy and anti-EcR. Hyaline haematocytes aggregated in layers below the wound site and granulocytes engaged in phagocytosis. Therefore, the immune system responds directly and rapidly to ecdysteroids. Epidermal cells developed EcR only several days after the haemocytes and only under intact carapace, not under the wound where they appeared apoptotic. At the wound margin, EcR-positive epidermal cells and fibroblasts proceeded to migrate across the wound between the layers of haemocytes. Epidermis was fully regenerated by day 15; at this time the ecdysteroid titre began rising towards a premoult peak and EcR disappeared from the nuclei of epidermal cells suggesting that high amounts of ecdysteroids exert negative control on EcR. When bacteria were injected at the time of wounding, both the plateau level of ecdysteroid titre and the cellular events of wound healing were prolonged by 5–7 days, showing that healing of the wound is slower and that the duration of the plateau phase of the titre depends on the degree of assault on the animal. We conclude that the low levels of ecdysteroids induced by wounding activate the immune system to begin healing below the wound and also stimulate adjacent epidermal cells to commence the process of wound repair.