The total duration of the moult cycle was measured as the interval between the two successive moults M1 and M2. Time between collection (i.e. assessment of the position in the moult cycle) and the first moult M1, relative to the total duration of the cycle, was used to

estimate the duration of the different phases of the moult cycle. Hence, we obtained a percentage of the cycle that was already completed per individual and average values were computed to obtain mean duration of each moult stage. The characterization of the selleck inhibitor moult cycle of G. pulex females followed the observation of a cyclic but constant and renewable phenomenon: the anatomical evolution of the dactylian claw and the dactylopodite shrinkage during a moult cycle. This has been previously described for other amphipods, Orchestia sp. and Niphargus virei (Charniaux-Cotton, 1957; Graf, 1968, 1986) and decapods (Drach, 1939; Drach & Tchernigovtzeff, 1967). For this purpose, the tip of the third right pereiopod was carefully cut off using fine INK128 forceps and gently placed in a drop of a Ringer solution

between a slide and a coverslip. Alternatively, the third left pereiopod and the right and left fourth pereiopods can be used for the dating of the same animal later in the moulting cycle. The claw of the pereiopod was observed under a Nikon Eclipse E600 microscope (×200 magnification). Pictures were obtained using a Nikon Digital Camera DXM1200F and software ACT-1 (Nikon, Tokyo, Japan). Pairs (amplexus) and unpaired males and

females were randomly sampled in the field (River Suzon) and placed into tubes filled with water. Collected animals were see more examined within 3 h. The moult stage was determined for all animals. Females were checked for embryos in the ventral pouch (resulting from a previous reproductive event). The type of moult of females (growth moult or egg-depositing moult) was also determined by checking the presence of maturing black ovaries, dorsally visible through the cuticle (vitellogenesis). We examined 138 pairs, 60 unpaired males and 52 unpaired females. Pairing status and behaviour according to female and male moult stages were analyzed with a χ2 test. Variation in size-assortative pairing was assessed overall using an analysis of covariance (ANCOVA) with the size of males as dependent variable and female size and moulting status as covariates. Pearson correlation tests were used to assess the magnitude of size-assortative pairing in each stage of the moulting cycle. All tests were performed using programs JMP© version 5 (SAS Institute, Cary, NC, USA). Females G. pulex with a size ranging from 1.68 to 2.58 (mean ± sd, 2.10 ± 0.19 mm) for the coxal plate (between 8.45 and 11.90 mm for the body length) have a 30-day moult cycle at 15°C (n = 44, mean 30.2 ± 3.6 days, range 24 to 40 days). It is subdivided into five periods accordingly (Fig.