Aminergic control of social status in crayfish agonistic encounters

Abstract

Using pairings of male crayfish Procambarus clarkii with a 3-7% difference in size, we confirmed that physically larger crayfish were more likely to win encounters (winning probability of over 80%). Despite a physical disadvantage, small winners of the first pairings were more likely to win their subsequent conflicts with larger naive animals (winning probability was about 70%). By contrast, the losers of the first pairings rarely won their subsequent conflicts with smaller naive animals (winning probability of 6%). These winner and loser effects were mimicked by injection of serotonin and octopamine. Serotonin-injected naive small crayfish were more likely to win in pairings with untreated larger naive crayfish (winning probability of over 60%), while octopamine-injected naive large animals were beaten by untreated smaller naive animals (winning probability of 20%). Furthermore, the winner effects of dominant crayfish were cancelled by the injection of mianserin, an antagonist of serotonin receptors and were reinforced by the injection of fluoxetin, serotonin reuptake inhibitor, just after the establishment of social order of the first pairings. Injection of octopamine channel blockers, phentolamine and epinastine, by contrast, cancelled the loser effects. These results strongly suggested that serotonin and octopamine were responsible for winner and loser effects, respectively.

Figure 1. Effect of physical size difference on crayfish agonistic bouts.

A, Winning rate of larger crayfish during agonistic bouts in pairings with smaller crayfish. The percentages of outcomes of agonistic bouts of larger crayfish are plotted as wins (black), losses (light grey) and draws (dark grey). Left: pairings between naive large (NL) and naive small (NS) animals, Middle: pairings between dominant large (DL) and dominant small (DS) animals, and Right: pairings between subordinate large (SL) and subordinate small (SS) animals. B, The time taken for the dominant-subordinate relationship to form. Box plots show median (solid black line), interquartile range (box length), and minimum and maximum values (error bars). Open circles indicate pairs that did not show clear dominant-subordinate relationship after 45 mins of pairings. C, The number of fights between pairings during the first 30 min agonistic bouts. D, The average duration of individual fights of pairings during the first 30 min of agonistic bouts. In C and D, the number of fights and the average duration of individual fights in subordinate pairings are separately plotted whether the dominant-subordinate relationship was determined or not. Asterisks in C and D indicate that the number and duration of responses differed significantly (Mann-Whitney rank sum test, *p<0.05, **p<0.01).

Figure 2. Effect of previous social experiences on agonistic bouts after 24 hrs.

A, Winning rate of crayfish during agonistic bouts. Dominant and subordinate crayfish were paired with un-experienced naive crayfish with a different body size on the next day. The percentages of winning outcomes of agonistic bouts of each group are plotted. From left to right: pairings between dominant large and naive small animals, dominant small and naive large animals, subordinate large and naive small animals, and subordinate small and naive large animals. No naive small crayfish paired with dominant large animals (Left) or subordinate small crayfish paired with naive large animals (Right) win. B, The number of fights of pairings during the first 30 min of agonistic bouts. C, The average duration of individual fights of pairings during the first 30 min of agonistic bouts. In B and C, the number of fights and the average duration of individual fights between dominant small and naive large crayfish are plotted separately whether dominant small animals win or lose. Asterisks in B and C indicate that the number and duration of responses differed significantly (Mann-Whitney rank sum test, *p<0.05, **p<0.01).

Figure 3. Effects of serotonin and octopamine upon agonistic bouts.

A, Winning rate of naive small crayfish injected serotonin at various concentrations 10-injected smaller crayfish are plotted. B, Winning rate of naive large crayfish injected octopamine at various concentrations 10 min prior to the pairings with naive small animals. The percentages of winning outcomes of agonistic bouts of octopamine-injected larger crayfish are plotted. Asterisks in A and B indicate that the winning rate of small crayfish (A) or large crayfish (B) differed significantly (Fisher’s exact test, *p<0.05). C, The number of fights of pairings during the first 30 min of agonistic bouts. D, The average duration of individual fights of pairings during the first 30 min of agonistic bouts. In C and D, data from saline-injected small (A) and large crayfish (B) are summed together as a saline group, and 1 and 2 µM serotonin-injected small crayfish are also summed. Asterisks in C and D indicate that the number and duration of responses differed significantly (Mann-Whitney rank sum test, *p<0.05, **p<0.01).

Figure 4. Effect of mianserin on agonistic bouts.

A, Winning rates of dominant small crayfish injected mianserin at various concentrations in pairings with naive large crayfish are plotted. Mianserin was injected into dominant animals immediately after establishment of dominant-subordinate formation, and then paired with naive large crayfish after overnight isolation. Asterisks indicate that the winning rate differed significantly (Fisher’s exact test, *p<0.05). B, The number of fights of pairings during the first 30 min of agonistic bouts. C, The average duration of individual fights of pairings during the first 30 min of agonistic bouts. In B and C, data of pairings between dominant small and naive large animals (Left) are from Fig. 2 that sum the data of both winning and losing dominant small crayfish. On the right, 20 and 50 µM mianserin injected into dominant small crayfish are also summed. The asterisk in C indicates that the number of fights differed significantly (Mann-Whitney rank sum test, **p<0.01). D, Effect of timing of mianserin injection into dominant small crayfish after establishment of social rank. The percentages of winning outcomes of agonistic bouts of mianserin-injected small crayfish are plotted. E, Effects of mianserin-injected naive large crayfish 1 hr or 10 min before pairing with naive small crayfish. The percentages of winning outcomes of agonistic bouts of mianserin-injected naive large crayfish are plotted as black bars and those of untreated naive small crayfish are plotted as grey bars.

Figure 5. Effect of phentolamine on agonistic bouts.

A, Winning rates of subordinate large crayfish injected with saline, phentolamine of 10 µM or 25 µM and 20 µM mianserin in pairing with naive small crayfish are plotted. Physiological saline, phentolamine or mianserin was injected into subordinate animals immediately after establishment of dominant-subordinate formation, and then paired with naive small crayfish after overnight isolation. B, The time during which the dominant-subordinate relationship was determined in both pairings between saline-injected subordinate large crayfish and untreated naive small crayfish and pairings between 25 µM phentolamine-injected subordinate large crayfish and untreated naive small crayfish. Box plots show median (solid black line), interquartile range (box length), and minimum and maximum values (error bars). Asterisk indicates that the time for social rank formation was different statistically (Log-rank test, *p<0.05). C, The number of fights of pairings during the first 30 min of agonistic bouts. D, The average duration of individual fights of pairings during the first 30 min of agonistic bouts. Asterisk indicates that the duration of responses differed significantly (Mann-Whitney rank sum test, *p<0.05).