The authors have declared that no competing interests exist.
Conceived and designed the experiments: LA PGG FG. Performed the experiments: LA AM CG. Analyzed the data: LA. Contributed reagents/materials/analysis tools: EF. Wrote the paper: LA.
Biogenic amines, particularly serotonin, are recognised to play an important role in controlling the aggression of invertebrates, whereas the effect of neurohormones is still underexplored. The crustacean Hyperglycemic Hormone (cHH) is a multifunctional member of the eyestalk neuropeptide family. We expect that this neuropeptide influences aggression either directly, by controlling its expression, or indirectly, by mobilizing the energetic stores needed for the increased activity of an animal. Our study aims at testing such an influence and the possible reversion of hierarchies in the red swamp crayfish,
Given the nearly ubiquity of social hierarchy across animal species (reviewed in
Crayfish are excellent model organisms to study the proximate mechanisms that invertebrates adopt to establish and maintain dominance hierarchies
The maintenance of stable hierarchies is certainly adaptive
To date, serotonin (5-HT, 5-hydroxytryptamine) is the main neuromodulator recognized to play an important role in controlling aggression in several crustacean decapods (the lobster
Crustacean HH is a member of a family of eyestalk neuropeptides
Notwithstanding the high number of papers on the cHH chemical nature, studies on its biological activity remain still scanty
To fill this gap in knowledge, here we investigate the possible influence that cHH exerts on the agonistic behaviour of the red swamp crayfish,
About 200 male crayfish were collected using baited traps from Lake Trasimeno (Umbria, central Italy) in July 2011 by professional fishermen. Once in the laboratory, each crayfish was individually marked onto its carapace with a waterproof paint and its cephalothorax length (from the tip of the rostrum to the posterior edge of the carapace) was measured using a vernier calliper (accuracy: ±0.1 mm).
Crayfish were kept for at least two weeks at the density of 15 m−2 in plastic tanks (80×60×60 cm) containing clay pots in excess as shelter and at a natural light-dark cycle at room temperature (24°C). They were fed
Only hard-shelled, intact, and sexually mature males were used for the experiment. A total of 80 individuals (cephalothorax length: 47.5±0.6 mm) were thus selected: 20 for the extraction of cHH and 60 for behavioural observations. Since dominance increases with body size in crayfish
Twenty animals were anesthetized for 5 min on ice before eyestalk ablation. From 40 eyestalks the crude extract of dissected sinus glands was collected by adding 200 µL of extraction solution (90% MetOH, 9% acetic acid, 1% H2O). After sonication, the sample was centrifuged at 12 000× g for 10 min at 4°C and the supernatant was collected. The pellet was suspended in 200 µL of the extraction solution, sonicated and centrifuged again, and the two supernatants were mixed together. The extract was purified on an RP-HPLC system (Gilson) equipped with a Zorbax SB-C18 4.6×150 mm column from Agilent Technologies Inc. (DE, USA) thermostated at 25°C. Mobile phase A was 0.1% TFA in water, mobile phase B was 0.1% TFA in acetonitrile. The separation was done using a gradient of 0–100% B in 60 min at 1 mL/min. The resulting chromatogram is shown in
Mobile Phase A: 0.1% TFA in water. Mobile Phase B: 0.1% TFA in acetonitrile. Gradient: 0–100% B over 60 min at 1 mL min−1. Column: Zorbax SB-C18 4.6 × 150 mm.
The collected fractions were analyzed on an API150EX single quadrupole mass spectrometer (ABSciex), and those fractions containing the expected molecular mass of 8386 Da
Behavioural experiments were conducted in the laboratory from 0800 to 1400 h during August 2011 to reduce possible interference due to circadian changes in blood glucose level
The animals were blotted dry and hemolymph (about 50 µl) was drawn from the pericardial sinus into sterile 1 mL syringes fitted with 25 g needles. All the animals were bled between 0800 and 0900 h and left undisturbed for 2 h. The sample was preserved on ice for 5 min to avoid coagulation and then centrifuged at 12 000× g for 2 min at 4°C to pellet the hemocytes. The supernatant was then collected. Glucose concentration (mg dL−1) in the hemolymph was assessed using the glucose oxidase method of a commercial kit (Hospitex Diagnostics).
The two opponents were kept in an experimental aquarium (a circular opaque PVC container, diameter: 30 cm) separated by an opaque PVC divider for 10-min acclimatization. The familiarization started with the removal of the divider and lasted 20 min (T0), during which time crayfish behaviour was recorded by a digital camera (Samsung VP-L800) for subsequent blind analysis (see below). Simultaneously, an experienced observer (LA) recorded the winner of each fight so that, at the end of familiarization, we could determine the dominant –alpha crayfish (and consequently the subordinate –beta crayfish) for each pair, that is, the winner (and the loser) of more than 60% of the total fights
The above selected pairs were randomly assigned to one of the following treatments: (1) ‘control pairs’ (CP, n = 8): both males were injected with 100 µL of phosphate buffered saline (PBS); (2) ‘reinforced pairs’ (RP, n = 9): the alpha was injected with 100 µL of native cHH solution, and the beta with 100 µL of PBS; (3) ‘inverted pairs’ (IP, n = 9): the opposite of the previous treatment.
To obtain cHH solutions, lyophilised native cHH was diluted with PBS to a final concentration of 5 µg mL−1, using 100 µL of such solution per individual, corresponding to 0.5 µg of cHH. The amount of the cHH injected into each crayfish was set from the results of a preliminary experiment showing that the injection of 0.5 µg of cHH determined a significant increase of the glycemic level in the hemolymph. Injections were made through the dorsal abdo-cephalothoracic membrane into the pericardial sinus using a 25 gauge × 5/8′ needle fitted to a 1 mL syringe.
Treated crayfish were left isolated and undisturbed for 30 min. Differently from serotonin and octopamine
The original pairs were reconstituted and observed following the same procedures as T0. After 10-min acclimatization, the divider was removed and crayfish behaviour was video-recorded 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.
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:
The number and total duration (in s) of fights. A fight began when one opponent approached the other and ended when one of the two individuals ran away, backed off or tail flipped away from the other for at least 10 s at a distance longer than one body length
Percentage of dominance (%). The number of fights won by an individual as a percentage over the total fights battled. The winner was the individual that did not retreat or that retreated after the opponent had 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
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
The fights started by alphas;
The time spent motionless (in s).
To compare variations of glycemia among treatments, immediately after the end of the experiment about 50 µL of hemolymph was drawn from each crayfish from 1300 to 1400 h, as described above.
Data 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
The experiments comply with the current 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).
As expected, the injection of cHH significantly increased glycemic levels in the crayfish hemolymph (F = 32.874, df = 2,52, P = 0.0001), independently of the hierarchical status of treated individuals (F = 0.0001, df = 1,52, P = 0.996). In fact, after cHH injections, glycemia significantly increased in a similar way (t = 0.57, df = 16, P = 0.995) in both the alphas of RP (t = −10.320, df = 16, P = 0.0001) and the betas of IP (t = 7.668, df = 16, P = 0.0001). Glycemic levels also increased in CP (in alphas: 17.6±6.3 mg dL−1, in betas: 16.6±3.2 mg dL−1) in response to fighting, but the recorded increment in the crayfish treated with cHH was about 10 times higher (alpha in RP: 178.6±20.4 mg dL−1; beta in IP: 177.1±15.0 mg dL−1).
Three asterisks denote significant differences at P<0.001 after Student's t-tests.
The total duration (F = 11.414, df = 3,69, P = 0.0001) and the number (F = 7.061, df = 3,69, P = 0.0001) of fights tended to decrease from T1 to T3 without any difference among treatments (F = 0.356, df = 2,23, P = 0.704 and F = 9.598, df = 6, 69, P = 0.748, respectively). As a consequence, the mean duration of fights was progressively shorter (F = 3.166, df = 3,69, P = 0.032) even if, immediately after the cHH injection, RP pairs combated longer than CP (F = 3.982, df = 2,25, P = 0.033; IP = CP<RP) (
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 (± 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.
One and three asterisks denote significant differences at P<0.05 and P<0.001, respectively, after Student’s t-tests.
Our 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 control individuals, 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. These behavioural changes were associated not only with an increased glycemia in the crayfish hemolymph, as well known in the literature
Our results show that cHH can modulate the neurons controlling the direct expression of agonistic behaviour also mobilizing the energetic stores needed for the increased fighting activity. Natural fluctuations of cHH release seem to be regulated by changes in central neuromodulation due to environmental and/or endogenous influences
Consistent with the study on the serotonin effects on
Undoubtedly, behavioural physiology opens new avenues for our understanding of the functioning of cHH and is expected to unravel its role in modulating invertebrate agonistic behaviour. Future researches are obviously needed to answer the exciting questions of how physiology and environment interact in regulating the neural systems underlying the formation and maintenance of social hierarchies across species.