End of the experiment (Fig. 4c). More often, fights were started

End of the experiment (Fig. 4c). More often, fights were started by the alphas (F = 5.765, df = 3,69, P = 0.001), although a difference was found with treatments (F = 3.571, df = 2,23, P = 0.045). While no difference was found between CP and RP, in IP the number of fights that the initial alpha started significantly decreased in T1 (F = 5.371, df = 2,25, P = 0.012; IP,CP = RP), as a consequence of cHH injections on betas. Alphas started a lower number of fights than betas also in T2 (F = 3.285, df = 2,25, P = 0.056) but this number tended to increase progressively through the experiment (F = 1.939, df = 2,25, P = 0.167) (Fig. 4d). Overall the time spent motionless increased with time (F = 2.992, df = 3,92, P = 0.035), with betas remaining motionless for longer than alphas (t = 22.355, df = 31, P = 0.025). The treatment had also an effect on activity (F = 3.594, df = 2,92, P = 0.031): cHH injections induced a more intense locomotion, so treated betas in IP spent the same time motionless as alphas (t = 20.355, df = 35, P = 0.725), whereas the different activity between treated alphas in RP and the respective beta increased (t = 24.797, df = 35, P = 0.0001) (Fig. 5).DiscussionOur study analysed the effects of cHH on the agonistic behaviour of crayfish and demonstrated, for the first time, its role in enhancing it, up to reverse, although transitorily, the rank. The supporting evidence is: as expected, (1) in CP and RP alphas increased dominance. On the contrary, (2) in IP, betas became likely to initiate and escalate fights and, consequently, increased dominance till a temporary reversal of the hierarchy, but the original rank was re-established. (3) In comparison with controlFigure 3. Difference in glycemic Mirin supplier levels after (1300?400 h) and before (0800?900 h) cHH injections (Mean+SE) in Control Pairs (CP), Reinforced Pairs (RP), and Inverted Pairs (IP), and in alphas (white bars) and betas (black bars). Three asterisks denote significant differences at 1676428 P,0.001 after Student’s t-tests. doi:10.1371/journal.pone.0050047.gAggression in Decapods Modulated by cHHFigure 4. Behavioural parameters across fighting bouts (T0, T1, T2, T3) in Control Pairs (CP), Reinforced Pairs (RP), and Inverted Pairs (IP). Before T0, the initial glycemia was determined; during T0, alpha and beta crayfish were assessed; between T0 and T1, crayfish were subject to the injection of either PBS solution (both alphas and betas in CP, alphas in IP, and betas in RP) or cHH solution (betas in IP and alphas in RP); from T1 to T3, crayfish behaviour was recorded and, then, the final glycemia was determined. Means (6 SE) of: (a) duration of fights; (b) percentage of dominance; (c) fight intensity level; (d) number of fights started by alphas. One and two asterisks denote significant difference at P,0.05 and P,0.01, respectively, after one-way ANOVAs. doi:10.1371/journal.pone.0050047.gindividuals, fights of treated alphas were longer and reached a higher intensity in treated betas. (4) IP betas showed reduced time spent motionless. To A 196 web summarize, independently of prior social experience, cHH injections induced expression of dominance that differs in relation to the original rank of the individual. TheseFigure 5. Mean time spent motionless (+SE) in Control Pairs (CP), Reinforced Pairs (RP) and Inverted Pairs 1662274 (IP), and in alphas (white bars) and betas (black bars). One and three asterisks denote significant differences at P,0.05 and P,0.001, respectively, after Student’s t-tests. do.End of the experiment (Fig. 4c). More often, fights were started by the alphas (F = 5.765, df = 3,69, P = 0.001), although a difference was found with treatments (F = 3.571, df = 2,23, P = 0.045). While no difference was found between CP and RP, in IP the number of fights that the initial alpha started significantly decreased in T1 (F = 5.371, df = 2,25, P = 0.012; IP,CP = RP), as a consequence of cHH injections on betas. Alphas started a lower number of fights than betas also in T2 (F = 3.285, df = 2,25, P = 0.056) but this number tended to increase progressively through the experiment (F = 1.939, df = 2,25, P = 0.167) (Fig. 4d). Overall the time spent motionless increased with time (F = 2.992, df = 3,92, P = 0.035), with betas remaining motionless for longer than alphas (t = 22.355, df = 31, P = 0.025). The treatment had also an effect on activity (F = 3.594, df = 2,92, P = 0.031): cHH injections induced a more intense locomotion, so treated betas in IP spent the same time motionless as alphas (t = 20.355, df = 35, P = 0.725), whereas the different activity between treated alphas in RP and the respective beta increased (t = 24.797, df = 35, P = 0.0001) (Fig. 5).DiscussionOur study analysed the effects of cHH on the agonistic behaviour of crayfish and demonstrated, for the first time, its role in enhancing it, up to reverse, although transitorily, the rank. The supporting evidence is: as expected, (1) in CP and RP alphas increased dominance. On the contrary, (2) in IP, betas became likely to initiate and escalate fights and, consequently, increased dominance till a temporary reversal of the hierarchy, but the original rank was re-established. (3) In comparison with controlFigure 3. Difference in glycemic levels after (1300?400 h) and before (0800?900 h) cHH injections (Mean+SE) in Control Pairs (CP), Reinforced Pairs (RP), and Inverted Pairs (IP), and in alphas (white bars) and betas (black bars). Three asterisks denote significant differences at 1676428 P,0.001 after Student’s t-tests. doi:10.1371/journal.pone.0050047.gAggression in Decapods Modulated by cHHFigure 4. Behavioural parameters across fighting bouts (T0, T1, T2, T3) in Control Pairs (CP), Reinforced Pairs (RP), and Inverted Pairs (IP). Before T0, the initial glycemia was determined; during T0, alpha and beta crayfish were assessed; between T0 and T1, crayfish were subject to the injection of either PBS solution (both alphas and betas in CP, alphas in IP, and betas in RP) or cHH solution (betas in IP and alphas in RP); from T1 to T3, crayfish behaviour was recorded and, then, the final glycemia was determined. Means (6 SE) of: (a) duration of fights; (b) percentage of dominance; (c) fight intensity level; (d) number of fights started by alphas. One and two asterisks denote significant difference at P,0.05 and P,0.01, respectively, after one-way ANOVAs. doi:10.1371/journal.pone.0050047.gindividuals, fights of treated alphas were longer and reached a higher intensity in treated betas. (4) IP betas showed reduced time spent motionless. To summarize, independently of prior social experience, cHH injections induced expression of dominance that differs in relation to the original rank of the individual. TheseFigure 5. Mean time spent motionless (+SE) in Control Pairs (CP), Reinforced Pairs (RP) and Inverted Pairs 1662274 (IP), and in alphas (white bars) and betas (black bars). One and three asterisks denote significant differences at P,0.05 and P,0.001, respectively, after Student’s t-tests. do.