2.3. Immune treatment
Immune treatment or sham-injection was administered 48 h after
the pre-treatment sampling of haemolymph, in order to let the octopuses
recover from haemolymph sampling (N = 35: 16 LPS-treated
and 19 controls). Among these 35 individuals, 12 LPS-treated animals
and 12 control animals were followed through the behavioural experiment
(see below, Behavioural experiment, and Fig. 1). The immune
challenge consisted of an intramuscular injection (1 ml/kg) of 15 mg/kg
of LPS (serotype: O26:B6) diluted 1.5% in phosphate buffered saline
(PBS). To establish this experimental dosage we made a preliminary test
just on some individuals with the injection of 5 mg/kg of LPS (from
literature on fish, see [11]). Since this dose did not elicit any change
of the immune parameters (i.e. number of circulating haemocyte and
serum lysozyme activity) we excluded it and concentrate on the higher
dosage of 15 mg/kg, that has been proved to elicit the activation of immune
system in other bivalve species [13,30]. Control animals were, at
the same time, sham-injected with an equal volume of PBS (1 ml/kg).
To perform the injection individuals were carefully encouraged to
leave their nest by showing them a crab from the surface and captured
using a hand-held net. One person gently held the octopuswhile another
proceeded with the injection. All individuals were injected with half