Biochemical energetics of hierarchy formation in Betta splendens

Physiology & Behavior, Vol. 43, pp. 447--450.Copyright©PergamonPress plc, 1988. Printedin the U.S.A.

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Biochemical Energetics of Hierarchy Formation in Betta splendens J. H A L L E R A N D C. W I T T E N B E R G E R Biological Research Center, 48 Republicii str. Ciuj-Napoca, R-3400, Romania R e c e i v e d 13 M a r c h 1987 HALLER, J. AND C. WI'I~rENBERGER. Biochemical energetics of hierarchy formation in Betta splendens. PHYSIOL BEHAV 43(4) 447--450, 1988.--Two different stages of hierarchy formation in Betta splendens were considered. Winners and losers in a short social contact, and dominant and submissive individuals after the establishment of a hierarchy, respectively, were identified. Metabolical determinations (free glucose, glycogen and protein content, glycogen and protein synthesis, glucose and amino acid oxidation, carbohydrate degradation) were performed. Winners and dominant individuals were shown to be able to produce more energy per unit time than losers and submissives, respectively. Differences in energy metabolism between individuals found in different stages of hierarchy formation also occurred: the carbohydrate degradation reached very high values after a short social contact. This is related to the noticed substitution of aggressive encounters with threat displays in the course of cohabitation.

Betta splendens

Social behavior

Hierarchy formation

Muscle metabolism

Energy metabolism

male Bettas are usually isolated in such small tanks, except for the breeding period, because of their aggressiveness). After this isolation period, two groups of 5 individuals were introduced into larger tanks (25x20x15 cm). Aggressive encounters were recorded, and after an hour the animals were sacrificed for metabolic determinations (test A). Two other groups of 5 individuals each were introduced in other, identical tanks and kept there for a week (previous observations made in the same tank on the same number of fisti showed that the hierarchy became stable within a week); after that they were sacrificed for metabolic determinations (test B). Finally, seven individuals served as controls; they were isolated (without the possibility of seeing each other) to the end of the experiment, when they were sacrificed. The temperature of the water in all tanks was 22-24 ° C. Water was cleaned periodically. The fish were fed on the small Annelid Tubifex sp., generally used as food for aquarium fishes. Test A was used to identify " w i n n e r s " and "losers" after a first short social contact. In common tanks, threat displays (erection of the gill covers and fins, changes in posture, [1,4]) appeared. If the threat display was returned, and none of the two involved individuals retreated, the displays resulted in fights (reciprocal attacks, bites and mouthlock [1, 4, 6]). The fish which retreated at the end of the encounter was considered a "loser;" its colour turned faint and it retreated in a corner of the tank for a while; the winner showed an intense colour and did not retreat. The total number of won fights was recorded for each fish, and, starting from these data, the individuals were classified into hierarchy-like rank orders. In each group, the first two individuals in the rank order were classified as " w i n n e r s " (w), the last two as "losers" (1). Thus, we obtained 4 winners and 4 losers. The results of metabolic determinations were grouped according to this classification.

H I E R A R C H Y formation and social status are generally related to physiological parameters such as hormones production rate, or circulating level of some hormones. It has been stated long ago that there is a correlation between plasma levels of sex steroids and social status [19]. It is known that in many cases "subordinate" status is related to an adrenal stress [7]. Some other papers extend their interest to a system other than the gonadal and adrenocortical ones, particularly to thyroid function [11,12]. Although the endocrine functions cited have strong metabolic effects, the correlation of social status with intermadiary metabolism has been studied less frequently. However, indirect evidence of this correlation exists: egg production in hens, growth in pigs, milk production in cows are related to social status [3], and all these depend to a great extent on metabolism. Generally it is accepted that the alpha status is very expensive; it requires a large investment of energy [16]. i n this paper, we want to search whether some relations exist between social status and energy producing ability of muscle tissue inBetta splendens. Bettas are considered to be highly territorial in natural habitats [5, 9, 13], but male and female adult individuals can establish a hierarchical system of social organization under aquarium conditions [6]. Many fish species, which are territorial in nature, form such social systems in captivity [10]. Therefore, we avoid generalizations which would refer to naturally social species, and yet we think that our results could extend beyond the species and experimental conditions studied. METHOD Twenty-seven young male Betta splendens, obtained from a local supplier (mean weight, 647_+96 mg; age, 6 months) were used for the experiment. All individuals were isolated in small tanks of 0.5 1 water volume for a week (the 447

448

H A L L E R AND W I T T E N B E R G E R

TABLE 1 THE RESULTSOF CALCULATIONSFOR SAMPLESIZE DECISIONS Group

w

1

V

P

0.48

99%

V

H P

0 . 0 2 99%

V

L P

0 . 4 8 99%

V

P

0.017 99%

In the case of H and L individuals "V" was calculated on the basis of the overall variance of the three measures of hierarchical order; P was found in a table given in [22]; for all V<0.5, P=99%.

In test B, we observed stable hierarchies. The social status of individuals was determined according to the following parameters: number of won fights per individual per hour, number of threat displays per individual per hour (those threat displays were counted which were not followed by fights, thus they were efficient in "intimidating" the opponent), competition for food. The result of competition for food was evaluated considering the number of Tubifex individuals captured from the total number of 25, administered for each meal. The methodology used in assessment of hierarchy orders was chosen based on indications found in the literature [4, 21, 22]. The a and fl individuals of each group (4 individuals) were classified as high social status individuals (H), the 3' and e individuals as low status individuals (L). The results of metabolic determinations were grouped according to this classification. The fish were killed by decapitation. The determinations were performed on the carcass of fishes, containing mainly muscle tissue, but also bones and skin. Total protein content of the carcass was determined by a gravimetric method based on precipitation with thrichloroacetic acid, purification with formic acid, and dehydration with acetone. Free glucose content was determined according to [17], glycogen content according to [15]. Carcass homogenates were incubated in labelled glucose containing buffered Krebs-Ringer solution (10 mM glucose, 2×105 DPM per ml with U-14C glucose, pH 7.4). The rate of glycogen synthesis (expressed as specific radioactivity DPM per mg glycogen) and the rate of glucose oxidation (expressed as CO2 radioactivity per mg tissue) were determined by observing the incorporation of radioactive carbon. In parallel fashion, the rate of carbohydrate degradation was determined according to the following formula: cd = gi + Gl - gt - Gf where g~ and G~ are the initial quantities of glucose and glycogen (before incubation), gf and Gf are the final quantities of glucose and glycogen of the incubated homogenates. A second incubation was performed with labelled leucine containing incubation medium (5 mM glucose, 2x 105 DPM per ml with 14C leucine, pH 7.4). The rate of protein synthesis (expressed as specific radioactivity, DPM per mg protein) and the rate of leucine oxidation (expressed as CO2 radioactivity per mg tissue) were determined following the incorporation of radioactive carbon. Incubations were performed in Warburg apparatus, radioactivity measurements were done in a BF-5003 liquid scintillation spectrometer (Berthold, Wildbad, Fed. Rep. Ger.) a PPO-POPOP mixture being used as scintillator. For the sample size decision we used the method described in [22]. This is based on the calculation of V [within

TABLE 2 MEASURES OF THE WINNERAND LOSER STATUS(TEST A), AND THAT OF SOCIALSTATUS(TEST B) Measures

No. of won fights No. of threat displays No. of worms captured

Test A

Test B

w

1

H

112+_12

12+_2.5"

13.5+_1.6

L 5+_0.5*

--

--

144+_32

21---6"

--

--

7.3-+0.8

3.1 ---0.2*

Results (mean+-SE) are given in number per individual per hour, and number per individual for "No. of worms captured," respectively. Asterisk indicates the statistical significance of differences (*/9<0.01). Number of individuals in each group is 4; w=the winners 1=the losers in test A, H=high social status individuals, L-low social status individuals in test B.

group variance (wgv)/between group variance (bgv)]. In our test B, where there were three measures of the social rank order, V was calculated as: l_~3(wgv) 1_~3(bgv) The chance to have a significant effect (P) was taken from the table given by [22]. The statistic evaluation of the differences between mean values of metabolic determinations was made according to the paired Student t-test. RESULTS The number of 4 individuals in w and l, H and L groups, respectively, was sufficient to characterize significantly these behavioral types in our experiment (see Table 1). The statistical calculus showed that the behavioral difference between w and l, H and L individuals, respectively, is significant (Table 2; even the losers have won some fights, because they were fighting also between them). The mean weight of w (714_+81.9 mg) and 1 (855_+95 rag) individuals has no significant correlation with the status of winner or loser. In test A, four of the eight metabolic parameters revealed statistically significant differences between w and l groups. The winners are characterized by a higher glycogen content of the carcass, a lower glycogen synthesis, increased carbohydrate degradation and slower leucine oxidation. If we compare the amount of oxidized glucose (Rgco2, Table 3) with the amount of oxidized leucine (Rlco~, Table 3), we find that I individuals burned 2 times as much leucine as glucose (p <0.02), while among winners the two rates of oxidation are similar (p >0.5). In test B, we obtained stable linear hierarchies in both groups of 5 individuals. The rank order of individuals was stable beginning with the 4th-5th day. In the course of the 7 days of cohabitation, the number of fights decreased progressively, while the number of displays increased (Fig. 1). All three measures of social rank orders gave the same rank

ENERGETICS OF HIERARCHY FORMATION

449

Nr./indJ h

TABLE3 THE ~SULTS OF METABOLICALDETER~NA~ONS Group g G cd RG Rgco2 P Rp Rlco2

w 1.06 _+0.11 2.95 -+0.18 10.3 -+0.44 514 -+84 0.32 -+0.06 102.2 •+14.9 7.8 _+1.01 0.46 -+0.02

l 0.91 -+0.05 1.71" -+0.29 7.75* -+0.58

10987 -+67 0.33 -+0.08 134.2 -+17.1 5.16 -+0.77 0.66?

-+0.04

H

L

C

1.32 -+0.16 2.89 -+0.4 2.17 -+0.3 895 -+125 0.34 -+0.01 138.5 -+8.5 1.49 -+0.34 0.67 -+0.17

0.71" -+0.15 1.89" -+0.08 0.83? -+0.2 531" -+77 0.30 -+0.04 109.7" -+9.5 0.56* -+0.11

1.01 -+0.16 2.37 -+0.35 4.32 -+0.33 432 -+71 0.34 -+0.06 111.0 -+4.4 1.02 -+0.22

0.65

0.45

-+0.28

-+0.06

Values (mean -- SE) are given in mg per g tissue for g (free glucose), G (glycogen) and P (proteins); in nag per g tissue per hour for cd (carbohydrate degradation); in DPM per mg of the given product for Rc (radioactivity of glycogen) and Re (radioactivity of proteins); in DPM per mg tissue for Rgco2 (radioactivity of CO2 in incubations with labelled glucose) and Rico2(radioactivity of CO2 in incubations with labelled leucine). ' T ' is compared with "w," " L " with "H." Statistical significance of differences (*p<0.05, ?p<0.01). Other explanations are given.

order. Mean weights of H (544___69 mg) and L (601___76 mg) individuals showed no significant correlation with social status. In test B, six of the eight metabolic parameters showed statistically significant differences between H and L groups. The H individuals were characterized by an increased glycogen content of the carcass, an increased free glucose content, increased carbohydrate degradation, increased glycogen sy__nthesis, increased rate of leucine incorporation into proteins, an elevated protein content of the carcass (Table 3). In some cases, the value obtained in metabolical determinations performed on the control group (C, isolated for the whole duration of the experiment) are between w and 1 and H and L values, respectively. These are the free glucose, glycogen and protein content of the carcass (Table 3). The degradation of carbohydrates in C reaches lower values than those of w and I individuals in test A, but is larger than those obtained in H and L individuals in test B. The rate of glycogen synthesis is similar to L and w individuals. The incorporation of leucine into proteins is comparable to that of H and L individuals, while oxidation of leucine is similar to that of w individuals and it is lower than that of 1, H, L, individuals (Table 3).

DISCUSSION The substitution of fights by threat displays in the course of cohabitation was also observed in experiments made in Hemigrammus acilifer [10]. While the aggressive encounters are progressively substituted by informational ones (threat displays), the total number of social contacts shows no significant changes. We consider that a community is socially stable, when the above-described situation has been established.

i

1

x

x

Attacks

i

I

i

I

I

i

2

3

4

5

6

7

days

FIG. 1. The change in the number of aggressive contacts (fights) and of informational ones (threat displays) in the course of the seven-day cohabitation.

We consider obvious that the social status reached by an individual must be in correlation with changes in energy metabolism, as it requires a large expenditure of energy [16]. Tests A and B reflect two stages of the same process: the formation of a stable hierarchical system in Betta splendens. Test A shows what kind o f modifications occur in the energy metabolism at the beginning of hierarchy formation, while test B shows what kind of modifications are stabilized in the course of cohabitation. The results prove that the winners are able to degrade carbohydrates more quickly, and that they are able t o produce more energy per unit time than the losers. Losers are mainly oxidizing amino acids. This could be a stress like result of losing. Although the rate of glycogen synthesis in losers is increased, and they degrade a lower amount of carbohydrates than winners, the glycogen content of their carcass is still low. Thus, the lower quantity of glycogen (energy) stores with which the individuals " s t a r t s " fighting could be one of the causes of loosing. It is probable that the lower velocity of carbohydrates degradation noticed in 1 individuals is also a cause of lost f~ghts. The results obtained in test B interfere with the situation which obtained in feeding: H individuals, because of their dominant position and greater success in competition for food, were better fed than L individuals, with the consequent increased protein content o f the carcass. The maintenance of a larger glycogen content of the carcass against the quicker carbohydrates degradation could be due also to these variation in foraging success. Thus, the increased rate o f glycogen and protein synthesis are consequences of the increased substrate availability. The values of carbohydrate degradation are much increased in all individuals in test A, and are much decreased in all individuals in test B. One can hypothesize that this is related to substitution of aggressive encounters with informational ones. In the first period of cohabitation, when a rank order is formed by energy expensive fights, the fish need much energy which is produced by degradation of carbohydrates. When the hierarchy is stable, and it is maintained by energy efficient threat displays the need for energy is lower, and the degradation of carbohydrates is slower. In

450

H A L L E R AND W l T T E N B E R G E R

the latter situation, we found increased amino acid oxidation, both in H and L individuals. It seems that in a group with a stabilized hierarchical system, the fish are sparing carbohydrates by oxidizing amino acids. This could be due to the low carbohydrate content of their food; it is known that amino acid oxidation is an important source of energy in carnivorous fish [23]. We shall further on try to compare our observations with the concepts of game theory elaborated by [24]; a review of the concept for sociobiology has been given by ([14]; see also [8]). In test A (where the individuals did not know each other) the result of a fight could not be foreseen. The value for which the individuals were competing was the priority for food (noted with P), as the quantity of food was limited; the cost of the competition (noted with C) is represented by the loss of energy in a physiological sense (a lowering of glycogen stores for example). The competition began with threat displays; there are two possible strategies: (1) retreat and (2) attack (nonretreating led immediately to attack). If the first strategy is chosen, the individual loses a priority ( - P ) . If strategy 2 (attack) is chosen the gain of the individual could be equal to + P - C , but it risks a loss equal to - P - C (if it loses the fight, both the priority, as well as the expended energy are lost). If P > C (as it must be, because otherwise the fights would be meaningless), the attack is the optimum strategy. In our experiment all the individuals chose the attack. The hierarchy once established (test B) the chance of an individual to gain something by attacking another one with higher status is very low. If it accepts the fight, he loses energy ( - C ) without the chance of gaining anything. On the other hand, high-ranking individuals are interested in

threatening, i.e., they reinforce the reached status and weaken the opponents (low-ranking individuals lose energy in escape swimming, which must be fast, otherwise they are attacked). In such a situation, the optimum strategy is the retreat in the face of individuals with higher social status, and the threat of individuals with lower status. Some conclusions might be drawn from the above statemeats: (1) the optimum strategies at the beginning and at the end of the hierarchy formation are different. (2) there is a gradual passage from the first optimum strategy (attack in every situation) to the second one [retreat in the face of dominant individuals and threatening the subordinate ones (see Fig. 1)]. (3) the hierarchy once established, low status individuals lose energy all the time, because they are permanently "hunted down" by individuals with higher status. This statement is supported by the marked biochemical differences between H and L individuals (test B) as compared to the differences noticed between winners and losers (test A). The permanent loss of energy in the case of the low ranking individuals, doubled by feeding difficulties, could lead to death within 4-5 weeks (Hailer, personal observation). (4) in the given experiment the game theory could be a useful concept in the explanation of phenomenons occurred (although some authors consider that the analysis of "strategies" played by individuals in groups are not very "instructive" [2]). We may conclude that there is a strong correlation between energy metabolism in B e t t a s p l e n d e n s and their social behaviour under aquarium conditions. To which extent our results can be applied to other species, which are social in natural habitats; and will be the subject of further research.

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