Use of X-ray micro-computed tomography to study the moult cycle of the freshwater amphipod Gammarus pulex

Stages of the moult cycle of the amphipod Gammarus pulex have been previously characterised based on the examination of either apolysis of the 3^rd dactyl, or the whole body and eye appearance. In the current study the aim was to compare these two established moult staging techniques with a novel X-ray micro-computed tomography (micro-CT) scan method. The micro-CT provides information on the degree of calcification of the external integument and of the internal structures, such as the gastric mill. The degree of calcification is predicted to change during the moult cycle. Successful micro-CT scans were obtained from 80 G. pulex specimens and the radiological appearance of the 28 specimens immediately immersed in 4 % PFA were not different to the 52 specimens stored in 4 % PFA for at least 28 days prior to scanning. These specimens could be classified into moult stages A, B, C, early D or late D based on the degree of calcification. Good agreement was obtained between all three methods of moult stage classification if fresh specimens were used, but if specimens had been preserved in 4% Paraformaldehyde (PFA) for more than 24 hours the loss of colour from the whole body and eye meant these methods were not suitable. This is the first time that a micro-CT method has been used to study G. pulex and shows that this method of moult staging is accurate and reliable.