Assumed a body down posture or remained motionless. As in familiarization

Assumed a body down posture or remained motionless. As in familiarization, the alpha was the individual that won more than 60 of the fights battled [40]; (3) Fight intensity (measured as the mode of the Acid Yellow 23 totalized scores). To each fight, classified as get Mirin avoidance, threat, week and strong physical interactions, and unrestrained fights (as modified from [4]), a score was assigned from 1 (avoidance) to 5 (unrestrained fight); (4) The fights started by alphas; (5) The time spent motionless (in s).Phase 5: Hemolymph sampling and determination of the final glycemia. To compare variations of glycemia amongtreatments, immediately after the end of the experiment about 50 mL of hemolymph was drawn from each crayfish from 1300 to 1400 h, as described above.Statistical AnalysesData were first checked for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene tests, respectively. All data met the assumptions for the parametric tests and were thus analysed accordingly. To test the effect of cHH injections on glycemia, we first computed the difference between glycemic levels after and before the injection, and then we applied a one-way ANOVA (statistic: F), in which the three treatments (CP, RP, and IP) and the two ranks (alpha and beta) were the between-subject factors and the difference in glycemic levels was the variable. The difference among treatments was explored by Tukey post hoc tests; in the case of significance, a comparison between alphas and betas was made by independent samples Student’s t test (statistic: t). To analyse the effect of cHH injections on the agonistic behaviour, we applied General Linear Models for repeated measures (GLMs, statistic: F), followed by Tukey post hoc tests, where the three treatments (CP, RP, and IP) were betweensubject factors and the four fighting bouts (T0, T1, T2, T3) were within-subject factors [42]. If the difference among bouts was significant, we applied one-way ANOVAs (statistic: F) to determine which treatments differed significantly. The level of significance is a = 0.05. The text reports means 6 SE.following the same procedures as T0. After 10-min acclimatization, the divider was removed and crayfish behaviour was videorecorded for three fighting bouts in sequence of 20 min each (T1, T2, T3). The experiment was timed to record the possible behavioural alterations due to cHH injections as a consequence of a major glucose release expected to occur in T2. In fact, from the literature (e.g. [34]) we know that cHH determines increased glycemia about 1 h after the injection. Videotapes were then blindly analysed by an unbiased observer (a PhD student), who was well experienced in crayfish behaviour but unaware of the experimental design and predictions. During T0 and the three fighting bouts we recorded:Aggression in Decapods Modulated by cHHEthical NoteThe experiments comply with the current 1326631 laws of Italy, the country in which they were done. No specific permits were required for the described field studies that did not involve endangered or protected species. The collection of animals did not affect the population density. Individuals were maintained in appropriate laboratory conditions to guarantee their welfare and responsiveness. After the experiments were completed, crayfish were killed by hypothermia because law forbids the release of invasive species into natural water bodies (L.R. 7/2005).Results Effect of Native cHH on Glycemia (Fig. 3)As expected, the injection of cHH sig.Assumed a body down posture or remained motionless. As in familiarization, the alpha was the individual that won more than 60 of the fights battled [40]; (3) Fight intensity (measured as the mode of the totalized scores). To each fight, classified as avoidance, threat, week and strong physical interactions, and unrestrained fights (as modified from [4]), a score was assigned from 1 (avoidance) to 5 (unrestrained fight); (4) The fights started by alphas; (5) The time spent motionless (in s).Phase 5: Hemolymph sampling and determination of the final glycemia. To compare variations of glycemia amongtreatments, immediately after the end of the experiment about 50 mL of hemolymph was drawn from each crayfish from 1300 to 1400 h, as described above.Statistical AnalysesData were first checked for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene tests, respectively. All data met the assumptions for the parametric tests and were thus analysed accordingly. To test the effect of cHH injections on glycemia, we first computed the difference between glycemic levels after and before the injection, and then we applied a one-way ANOVA (statistic: F), in which the three treatments (CP, RP, and IP) and the two ranks (alpha and beta) were the between-subject factors and the difference in glycemic levels was the variable. The difference among treatments was explored by Tukey post hoc tests; in the case of significance, a comparison between alphas and betas was made by independent samples Student’s t test (statistic: t). To analyse the effect of cHH injections on the agonistic behaviour, we applied General Linear Models for repeated measures (GLMs, statistic: F), followed by Tukey post hoc tests, where the three treatments (CP, RP, and IP) were betweensubject factors and the four fighting bouts (T0, T1, T2, T3) were within-subject factors [42]. If the difference among bouts was significant, we applied one-way ANOVAs (statistic: F) to determine which treatments differed significantly. The level of significance is a = 0.05. The text reports means 6 SE.following the same procedures as T0. After 10-min acclimatization, the divider was removed and crayfish behaviour was videorecorded for three fighting bouts in sequence of 20 min each (T1, T2, T3). The experiment was timed to record the possible behavioural alterations due to cHH injections as a consequence of a major glucose release expected to occur in T2. In fact, from the literature (e.g. [34]) we know that cHH determines increased glycemia about 1 h after the injection. Videotapes were then blindly analysed by an unbiased observer (a PhD student), who was well experienced in crayfish behaviour but unaware of the experimental design and predictions. During T0 and the three fighting bouts we recorded:Aggression in Decapods Modulated by cHHEthical NoteThe experiments comply with the current 1326631 laws of Italy, the country in which they were done. No specific permits were required for the described field studies that did not involve endangered or protected species. The collection of animals did not affect the population density. Individuals were maintained in appropriate laboratory conditions to guarantee their welfare and responsiveness. After the experiments were completed, crayfish were killed by hypothermia because law forbids the release of invasive species into natural water bodies (L.R. 7/2005).Results Effect of Native cHH on Glycemia (Fig. 3)As expected, the injection of cHH sig.

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