After acclimatization haemolymphwas sampled from each individual
(N = 35) to measure the baseline values of immunological parameters,
i.e. number of circulating haemocytes (N = 34) and serum lysozyme activity
(N = 28), before immune-treatment (T = 0 h). For haemolymph
collection individuals were carefully encouraged to leave their nest by
showing them a crab from the surface and captured using a hand-held
net. They were then anaesthetized through immersion in a solution of
55 mM MgCl2 + 1% ethanol in sea water for 15 min [24]. Haemolymph
(70–100 μl) was sampled from the branchial hearts (alternatively from
right or left heart in successive samplings) using a 2.5 ml syringe with
30G needle. The needlewas always inserted for less than half of its length,
paying attention that the insertion pointwas alwaysmedial. This allowed
the needle to reach the middle part of the branchial heart where space
is free and thus the risk of contamination with other cell material from
the heart soft tissue is extremely reduced. Furthermore the haemolymph
cell counting (see Section 2.4 below) allowed us to exclude the contamination
of haemolymph with undesired tissue.