Changes in the darkness of four body features of bluegill sunfish (Lepomis macrochirus Rafinesque) during aggressive encounters

BEHAVIORAL BIOLOGY, 14, 41-49 (1975), Abstract No. 4244

Changes in the Darkness of Four Body Features of Bluegill Sunfish (Lepomis macrochirus Rafinesque) During Aggressive Encounters PETER STACEY and DAVID CHISZAR Department of Biology, Department of Psychology, University of Colorado, Boulder, Colorado 80302 The darkness of four body features (general body surface, GBS; lateral stripe pattern, LSP; opercular flap spot, OFS; and dorsal tin-ray spot, DFRS) was observed during the formation of 16 pairwise status relationships among bluegill sunfish (Lepornis macrochirus Rafinesque). In eight cases (Method A) an intruder fish was placed into a 110 liter aquarium occupied for 7 previous days by a resident fish. In eight additional cases (Method B) two fish lived on opposite sides of a 110 liter aquarium divided by an opaque screen for 7 days prior to removal of the screen. Dominance was established by one of the fish in all but two cases (both were Method B) and aggressive encounters proceeded through five clearly identifiable stages: (1)a stage of inactivity just after pair formation, (2)a stage in which the fish oriented and approached each other tentatively and without further interaction, (3) a stage in which threat displays are emitted, often by both fish, but no actual body contact occurs, (4) a stage of vigorous attacks, and (5) a stage where one fish is clearly emitting all attacks and where the other fish is fleeing and/or hiding. Darkness of each feature in each fish was evaluated with a statistically reliable three-point scale at each stage during the contest. GBS increased in darkness equally for dominant and submissive fish. LSP, OFS and DFRS became darker in dominant fish and lighter in submissives, suggesting that these three features may have status-related signal value. Method of pair formation did not interact with these effects. The ability to change color, pattern, a n d / o r brightness is a widespread p h e n o m e n o n a m o n g teleost fishes, and these changes are k n o w n or suspected to serve m a n y adaptive functions (cf. Waring, 1963). Perhaps the most often cited e x a m p l e o f a color-change effect in the ethological literature is the role of the red belly in the male three-spine stickleback in indicating sexual readiness to females and in eliciting aggression by other territorial males (Tinbergen, 1951; but see Peeke, Wyers and Herz, 1969). Change o f colors a n d / o r brightness patterns during the course o f aggressive encounters has been observed in m a n y fish species, but systematic studies o f this p h e n o m e n o n are relatively rare in the literature. The w o r k o f Baerends, et al. (1955) w i t h 1Contribution number 62 from the Laboratory of Animal Physiology and Behavior, University of Colorado, Boulder, CO. 41 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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guppies (Lepistes reticulatus), Barlow (1963) with the Asian teleost Badis badis, and Niel (1964) with Tilapia mossambica has indicated that such changes can often be correlated with the sequential events of aggressive interactions; and, as Barlow (1963, p. 97) points out: "Color changes, or the existing color pattern, frequently indicate motivational states in the absence of other overt behavior, or they may constitute an additional source of information . . . . " Sunfish of the genus Lepomis have been reported to exhibit changes in body brightness subsequent to dominance contests. Submissive green sunfish (Lepomis cyanellus) become conspicuously darker than dominants (McDonald and Kessel, 1967; McDonald, Heimstra and Damkot, 1968). The same correlation between body brightness and status has been routinely observed for bluegill sunfish (L. macrochirus) in our laboratory (Corey, 1974). However, it must be emphasized that the relative darkness of the submissive individuals appears after the dominance contest has been conclusivel3/ settled and after the submissive fish has been repeatedly attacked. This may require several days of enforced cohabitation. In fact, increasing darkness of the body is often a sign of tissue damage in bluegills. Accordingly, the darkness of the submissive fish probably represents a terminal deferential condition rather than a signal which is functional in the initial establishment of status relationships. Indeed, several observers in our laboratory have indicated that during the decisive interactions between co-habitants, which usually occur within the first hour, several aspects of the submissive individual's body actually become lighter than comparable areas of the dominant's body. Figure 1 presents a drawing of a bluegill and shows three major melanophore-containing features: (1)the opercular flap spot (OFS), (2)the dorsal fin-ray spot (DFRS), and, (3)the lateral stripe pattern (LSP). Each of these features appears to be especially conspicuous in dominant individuals and relatively inconspicuous in submissives; and, these differences are usually present within an hour after two fish have been placed together (i.e., long before the submissive begins to darken). The purpose of the present study is to measure the changes in these features during various kinds of aggressive encounters in order to verify the correlation between feature appearance and status, and to provide information which could lead to the construction of appropriate models to test the signal value of various anatomical attributes.

METHOD

SubJects. The bluegills (8.5-13.0 cm fork length) used ill this study were seined from a local pond and were maintained in a large (2000 liter) tank at approximately 18-+ 2°C for 1 mo prior to the start of the experiment. The laboratory light period was from 0700 to 1900 and fish were never disturbed

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Fig. 1. Outline of a bluegill sunfish (Lepomis macrochirus) showing the melanophore-containing areas measured in this study. OFS-opercular flap spot, a relatively small but distinct area of about 0.7 cm2 DFRS-dorsal fin-ray spot, located in the posterior fin rays and when fully dark may have an area close to 1.0 cm2 in large animals. LSP-lateral stripe pattern; it contains five to eight parallel bars which can vary in the extent to which they traverse the entire side. LSP may be completely absent or the bars may extend completely to the ventral margin of the body. In addition to these three features, the general body surface (GBS) may vary in darkness. In rating GBS, the head is ignored since it contains relatively few melanophores but some chromatophores. at night. Herring-meal pellets and/or mealworm larvae of Tenebrio molitor were offered once daily between 1200 and 1400 and all fish accepted these foods readily. Experimental Conditions. Two methods of pair formation were employed. In Method A a single bluegill was transferred from the stock tank to a 110 liter aquarium (at 18-+2°C) equipped with charcoal filter and aerator. This individual, now called a resident, was allowed to remain undisturbed (except for the introduction of food) in this aquarium for seven days. Isolating the fish in this manner virtually insures that a conclusive dominance contest will occur when a second fish of equal size (the intruder) is added to the aquarium. The pair was continuously observed until a clear dominance relationship was established as judged by the emission of attacks exclusively by one of the fish. In Method A the resident always emerges as the dominant fish and attacks usually appear between 30 min and 1 hr after introduction of the intruder. A total of eight resident-intruder pairs were observed. In Method B a 110 liter aquarium was divided into two equal volume compartments by an opaque screen and equal sized bluegills were placed in the respective compartments. The screen was removed after seven days and the pair was continuously observed until the dominance criterion described above was reached. Method B usually generates more intense aggressive behavior by both fish and the dominance contest may last several hours. In fact, clear dominance relations sometimes do not emerge. Eight pairs of bluegills were observed with Method B and two of these were discarded because clear status differences did not become apparent within 10 hr.

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Dependent Variables. A three-point scale was used to rate increasing darkness of: (1) general body surface, (2) DFRS, (3) OFS and (4) LSP. For all features except LSP, a score of 1, 2, or 3 represented light or pale, medium and dark, respectively. For LSP, a score of 1 was assigned if no stripes were visible, a score of 2 was assigned if stripes extended only part way down the body, and a score of 3 was assigned if a full stripe pattern was present. Six preliminary observation sessions were devoted to the determination of inter-rater agreement for the use of the three-point scale. Two observers assigned scale values (for all four features) to each fish of these six pairs at ten points during the respective encounters. Chi squares comparing observed frequency of agreement with chance expectation were calculated for each feature in each fish and then pooled across fish (Snedecor, 1946). All values were significant (all Ps < 0.001), indicating excellent inter-rater agreement in use of the three-point scales to rate darkness of body features; and, percentage agreement did not differ significantly from fish to fish o r from feature to feature. Sampling. A dominance contest in bluegills typically proceeds through five stages which can be characterized in terms of the behaviors which are most prevalent. Stage 1 (called Start) occurs when the fish are initially placed together and is marked by the total absence of interaction. The individuals usually have elevated respiration rate, reflecting the fact that they have been disturbed (Chiszar, Moody and Windell, 1972). Within 5-10 rain the fish begin to orient and approach each other and this constitutes Stage 2 (called Approach). Stage 3 (called Circle and Display) results when mutual lateral and/or frontal displays appear (Miller, 1964). Often the fish appear to circle each other as they attempt to maintain reciprocal displays. Next, biting attacks emerge (i.e., Stage 4, called Attack). In Method A, it is always the resident who initiates attacks; in Method B, both individuals may exhibit this behavior, at least initially. Eventually one fish initiates all attacks and the other flees and/or attempts to keep himself concealed behind airlines, stones or in an upper corner of the aquarium. This is Stage 5 (Dominance Established) or the point at which clear status relationships are visible. Role reversals almost never occur, even if the pair is left intact for weeks. Although the five stages can vary in duration, observers never disagree in identifying them. Because of the reliability of this index, it was used as the basis for sampling darkness values. All four features of each fish were rated during each of the five stages. Often individuals received several ratings during each stage, and, in these cases the modal rating was employed in subsequent analyses. RESULTS Figure 2 present mean scale values as a function of stage for dominant and submissive individuals observed with both methods of pair formation.

SUNFISH BODY FEATURES DURING AGGRESSION MET -IOD

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Fig. 2. Mean darkness ratings for GBS, LSP, OFS and DFRS are plotted as a function of five successive stages of the aggressive encounter. Data are plotted separately for dominant and submissive fish, and for both methods of pair formation.

Data for each feature were subjected to a 2 × 2 X 5 mixed ANOVA where factors were Method, Status and Stage. Since two pairs of fish in Method B failed to form clear status relationships, they were dropped from the analysis and they were not included in Fig. 2. Accordingly, ANOVA solutions were obtained using the unweighted means procedure. Table 1 summarizes all ANOVAs.

46

STACEY AND CHISZAR TABLE 1 Summary of Unweighted-Means ANOVAs for Each Feature General body surface Source of variation

df

Method Status Method X Status Stage Method × Stage Status X Stage Method × Status X Stage

1,24 1,24 1,24 4,96 4,96 4,96 4,96

Lateral stripe pattern

Opercular flap spot

Dorsal Finray spot

F ratios

1.05 0.42 0.16 12.87"* 0.62 0.83 0.62

5.57* 1.28 0.09 14.78"* 2.34 2.52* 0.99

5.88* 7.58** 0.60 9.92** 3.53** 7.06** 1.19

3.78 2.85 0.42 10.00"* 5.49** 8.02** 1.91

*P < 0.05. **Y < 0.01. Significant main effects of Stage were obtained for all features, indicating that darkness increased during agonistic encounters. However, the Stage effect interacted with Status for LSP, OFS, and DFRS. Although the Status main effect was significant only for OFS, it is clear that for all measures (except GBS) the submissive fish in both Methods were lighter than dominants in the later stages, particularly Stage 5. Hence, LSP, OFS and DFRS change differentially depending upon status and, therefore, these are the features which are most likely to have signal value. Since rating of GBS did not depend upon status, there is no reason to suspect this feature to function as an indicant of dominance or submissiveness. Although detailed measures of aggressive behavior were not taken in this experiment, it was only in Method B that actual dominance contests occurred. Hence, frequency o f attacks was probably higher in this case. It is not surprising, therefore, that main effects of method were observed for LSP and OFS (the method main effect for DFRS was marginally significant: 0 . 1 0 > P > 0 . 0 5 ) . This is largely a result of the fact that in Method B the submissive fishes darken as the encounter progresses until status relationships are finally decided. However, it is important to recognize that no triple interaction was significant, indicating that the magnitude of the Status X Stage effect does not depend upon method of pair formation. DISCUSSION The occurrence of color or brightness change during behavioral episodes is not sufficient evidence of signal function. Two additional kinds of

SUNFISH BODY FEATURES DURING AGGRESSION

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information are necessary: (1)the morphological changes must be shown to correlate with some short- or long-term behavioral-outcome criterion; and (2) the morphological changes must be capable of independent manipulation in order to assess their effects in a context where the respective functions of morphology and behavioral concomitants can be separated (e.g., modeling experiments). The present investigation reveals that at least three aspects of the bluegill body exhibit differential change in darkness in a manner which is correlated with status: LSP, OFS and DFRS became clearly darker and more conspicuous in dominants and paler in submissives. In fact, these features may disappear completely from submissive fish. Accordingly, it seems reasonable to hypothesize that all three of these features may advertise status; increasing darkness may also exert intimidating effects on cohabitants while paleness may be differential. Bluegills and several other species of sunfish form stable dominance hierarchies in the laboratory (Erickson, 1967), particularly when more than two individuals are placed together. Similar behavior may occur in nature during the non-reproductive season in cases where individuals are forced to aggregate because of food concentration or drying up of sections of the habitat. Perhaps the darkness changes demonstrated in this study play a role as status signals in the formation and/or maintenance of hierarchical systems. Status signals may also play an important role in the reproductive behavior of sunfish. During the breeding season, males build and defend nests (Avila, 1973; Miller, 1964; Morgan, 1951). Females, however, do not occupy territories; rather, they briefly enter the male's nest to spawn. Although both sexes appear capable of developing dark LSP, OFS and DFRS in the laboratory, territorial males are typically the only subjects with dark features under natural reproductive conditions. It can be hypothesized that these dark features advertise the presence of a territorial male while the paler condition indicates a reproductively motivated female. Model experiments are currently being conducted to determine the kind and extent of behavioral changes consequent to presentation of silhouettes with different combinations of LSP, OFS and DFRS. It is noteworthy that only one feature, OFS, exhibited extremely rapid change. This spot could disappear within 2 or 3 sec whereas all other features changed only gradually, requiring many minutes for complete darkening or lightening. Since it is generally believed that rapid color changes are neurally controlled while slower changes are hormonally controlled (Knowles, 1959), it is likely that the OFS may be mediated by different factors than the other features measured in this study. This suggests that the OFS may have behavioral effects which differ from those of the other more slowly acting features. If a pair consisting of a dominant and a submissive bluegill is allowed to continue cohabiting an aquarium after the status relationships are formed, the

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submissive often sustains many attacks and its body becomes extremely dark. In fact, the darkness of its body completely obscures visibility of the stripes as well as the relatively small OFS and DFRS. Hence, total darkening of the body may represent a long-term adjustment to the chronic stress of violent domination. However, such chronic stress probably also generates severe hormonal alterations which could produce the melanistic condition as a non-functional side effect (e.g., corticotrophins are known to have melanogenic effects; Turner, 1960). Definitive modeling experiments are required to test these ideas. In either case it is important to recognize that the darkness of submissive bluegills is a "terminal" condition and that various distinct body features undergo additional changes in brightness during early stages of aggressive encounters. Accordingly, emphasizing only the long-term darkening process ignores the possible communicatory roles of short-term changes in LSP, OFS and DFRS.

REFERENCES Avila, V. L. (1973). A review and field study of nesting behavior of male bluegill sunfish (Lepomis macrochirus Rafinesque). Ph.D. thesis, University of Colorado, Boulder, pp. 108. Baerend, G., Brouwer, R., and Waterbolk, H. (1955). Ethological studies on Lebistes retieulatus (Peters). Behaviour 8, 249-334. Barlow, G. (1963). Ethology of the Asian teleost Badis badis. III. Motivational and signal value of the color patterns. Anita. Behav. 11, 97-105. Chiszar, D., Moody, M., and Windell, J. T. (1972). Failure of bluegill sunfish, Lepomis macroehirus, to habituate to handling. J. Fish Res. Bd. Canada 29, 576-578. Corey, C. (1974). Interspecific and intraspecific aggression in Lepomis gibbosus and L. macrochirus. J. Colo.-Wyo. Acad. Sci. 7, 7. Erickson, J. T. (1967). Social hierarchy, territoriality and stress reactions in sunfish. Physiol. Zool. 40, 40-48. Knowles, F. G. W. (1959). The control of pigmentary effectors. In A. Gorbman (Ed.), "Comparative Endocrinology." New York: John Wiley & Sons. McDonald, A. L., Heinlstra, N. W., and Damkot, D. K. (1968). Social modification of agonistic behavior in fish. Anirn. Behav. 16, 437-441. McDonald, A. L., and Kessel, L. A. (1967). Relationship between social hierarchy and coloration in green sunfish. Psychol. Rep. 20, 748-750. Miller, H. C. (1964). The behavior of the pumpkinseed sunfish Lepomis gibbosus (L.), with notes on the behavior of other species of Lepomis and the pigmy sunfish, Elassoma evergladei Behaviour 22, 88-151. Morgan, G. D. (1951). The life history of the bluegill sunfish, Lepornis rnaeroehirus, of Buckeye Lake, Ohio. Denison University Bull. J. o f Sci. Lab. 42, 21-59. Niel, E. H. (1964). Color patterns in Tilapia mossambica. Un. Cal. Pub. Zoo. 75, 1-58. Peeke, H. V. S., Wyers, E. J., and Herz, M. J. (1969). Waning of the aggressive response to male models in the three-spined stickleback (Gasterosteus aeuleatus L.). Anirn. Behav. 17, 224-228. Snedecor, G. W. (1946). "Statistical Methods" (4th Ed.). Ames, IA: Iowa State College Press.

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Tinbergen, N. (1951). "The Study of Instinct." Oxford: Oxford University Press. Turner, C. D. (1960). "General Endocrinology," 3rd ed., pp. 74-78. Philadelphia: W. B. Saunders Co. Waring, H. H. (1963). "Color Change Mechanisms of Cold Blooded Vertebrates." New York: Academic Press.