Organization of motor and posture patterns in paradise fish ...

Behavior Genetics, Vol. 25, No. 4, 1995

Organization of Motor and Posture Patterns in Paradise Fish (Macropodus opercularis): Environmental and Genetic Components of Phenotypical Correlation Structures R o b e r t G e r l a i 1,2 a n d W i m E. C r u s i o 3

Received 21 July 1994-Final 11 Jan. 1995

Paradise fish exhibit complex, environment-specific behavioral responses which consist of behavioral elements (motor and posture pattems) appearing in a typical, correlated manner. The genetic and environmental components tmderlying these phenotypical correlations have not been comprehensively investigated. Therefore, we have analyzed the behavioral elements of paradise fish from the nine populations of a 3 X 3 full diallel cross by employing a bivariate extension of the Hayman-Jinks variance-covariance analysis, demonstrating the presence of significant environmental and genetic correlations. To investigate the multivariate structure of the correlation matrices obtained, we subjected the phenotypical, environmental, additive genetic, and dominance correlations to principal-component analyses (PCAs). After rotation, the phenotypical principal factor pattem found was similar to previously obtained ones, suggesting stable underlying biological mechanisms. The environmental PCA extracted several environmental principal factors that were highly situation-specific. PCAs of the matrices of genetic correlations extracted only a small number of genetic principal factors which were not situation-specific, suggesting a relatively simple underlying genetic structure. K E Y W O R D S : Paradise fish; behavioral element; diallel cross; genetic correlation; behavioral organization; multivariate analysis.

INTRODUCTION

W e chose paradise fish as our m o d e l s y s t e m since this species exhibits a rich behavioral repertoire that reflects a c o m p l e x behavioral organization. W e utilize the ethological m e t h o d o f recording several spontaneously emitted behavioral responses. The application o f ethological m e t h o d s in behavior-genetic analyses was p i o n e e r e d m o r e than 30 years ago b y v a n A b e e l e n (1963), w h o stated that " f o r a fiu'ther d e v e l o p m e n t o f b e h a v i o u r genetics it s e e m s desirable that b e h a v i o u r should be studied in all its multiformity; in this respect behaviour genetics m a y greatly profit f r o m ethological attainments and procedures, a m o n g which the drawing up o f ethograms, i.e. b e h a v i o u r inventories, c o m e s first." With the help o f an ethogram, s e e m i n g l y continuous b e h a v i o r is described as a se-

In this p a p e r we analyze the behavioral organization o f paradise fish (Macropodus opercularis) with multivariate genetic m e t h o d s and investigate h o w genetic and environmental correlations give rise to an observable phenotypical correlation pattern, a question that has not b e e n studied before. 1 Department o f Ethology, L. E r t v r s University o f Budapest, G6d, Jfivorka S. u. 14, 2131 Hungary. 2 To whom correspondence should be addressed at Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Neurobiology Division, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. Fax: (416) 586-8588. e-mail: [email protected]. s G~n&ique, Neurog~n&ique et Comportement, U R A 1294 CNRS, UFR Biomrdicale, Universit6 Paris V, 45 Rue des Saints-Pbres, 75270 Paris Cedex 06, France.

385 0001-8244/95/0700-0385507.50/0 9 1995 PlenumPublishingCorporation

386

quence of successive, mutually exclusive and distinct motor-posture patterns that represent speciesspecific units of behavior which may be subsequently quantified (see, e.g., Huntingford, 1984). Although mutually exclusive and distinct, these units appear in an organized, correlative manner, which may be investigated by multivariate statistical methods such as principal-component analysis (PCA). The paradise fish is a small, 10-cm-long, freshwater species from Southeast Asia that exhibits a rich behavioral repertoire involving several motor and posture patterns, the behavioral elements (Cs~inyi et al., 1985a, b). These behavioral elements are discrete units of behavior (Csfinyi and Gerlai, 1988), which, however, do not appear independently of each other. Depending on the environmental circumstances, they form specific, phenotypically correlated behavioral clusters (Gervai and Csfinyi, 1985; Gerlai and Csfinyi, 1990). These clusters have been interpreted as resulting from underlying biological mechanisms reflecting behavioral states (see, e.g., Csfinyi et al., 1985a; Gervai and Csfinyi, 1985; Gerlai and Csfinyi, 1990). Although such a behavioral interpretation allows one to visualize a complex behavioral response, it does not render any information about the multivariate structure of the underlying environmental and genetic effects. Elucidating this structure is one of the aims of the present study. One may study how genotype and environment influence the correlated appearance of behavioral elements by manipulating both factors, i.e., by observing animals of different genotypes in a variety o f experimental situations. Since we did not have any data on how specific environmental cues might affect the interrelationships of behavioral elements, we did not attempt to manipulate systematically single environmental stimuli. Instead, we chose to apply a number of complex experimental situations (see Gerlai and Csfinyi, 1990) that we assumed would elicit different behavioral responses. These situations were designed to model certain features of the natural habitat of paradise fish (Gerlai et al., 1990). We applied a widely used quantitative-genetic design, the diallel cross, to dissect out environmental and genetic effects. The diallel cross comprised three inbred strains of paradise fish and all possible F1 hybrid generations among them. This crossbreeding design allowed a detailed analysis of the

Gerlai and Crusio

genetic underpinnings of a phenotype (Hayman, 1954; Jinks and Hayman, 1953) and the bivariate extension of this analysis permitted a multivariate inquiry into the observed multiformity of behavior (see Crusio, 1992, 1993). The great degree of environmental differences between the applied test situations and the large genetic differences between the inbred strains used in this study (Csfinyi et al., 1985a; Gerlai et al., 1990) facilitate revealing possible covariations between behavioral elements, be they of environmental or genetic in origin. Multivariate analysis of these covariations allows us to investigate the mode of contribution of environmental and genetic effects to the observable phenotypical correlation structure.

METHODS Animals and Housing The inbred lines of paradise fish used in this study, U, P, and C, were founded in 1978 and had been sib-mated for 22 generations, resulting in a high level of inbreeding by the time of testing. They originated from different sources in different countries and we may assume that they were already genetically different at the start of the inbreeding. Although no deliberate, divergent artificial selection was employed, the lines presumably had become genetically even more distinct because of random fixation of different alleles (i.e., genetic drift). Their behavioral and genetic distinctiveness made them appropriate as parental generations for the diallel cross. The diallel cross was comprised of the three parental inbred lines and all possible F1 hybrid generations among them. F 1 hybrids are indicated by a combination of the letter symbols of the parental lines, with the maternal parent mentioned first. All nine populations of the diallel cross were bred simultaneously so that the experimental fish were of the same age, 4-5 months, at the start of behavioral recording. Each population of the diallel cross was a brood of one pair of fish. All fish were raised and kept under identical conditions in heavily filtered 80-liter water tanks (30 fish per tank) at a temperature of 28~ and a 14/10-h light/dark cycle. The tanks contained a plant, Hygrophila polysperma, from the natural habitat of paradise fish. The fish were fed once a day on a calcium- and vitamin-

B e h a v i o r a l O r g a n i z a t i o n in Paradise Fish

enriched artificial fish food consisting o f beef liver, hake, eggs, wheat bran, and tuna fish. In the behavioral tests only females were used, since males exhibit higher behavioral variability as a result o f both their territoriality and possible differences in dominance hierarchy status (personal observation). Behavioral Testing

The fish originating from the nine populations of the diallel cross (U, n = 20; UP, n = 21; UC, n = 2 9 ; P U , n = 2 1 ; P , n = 20; PC, n = 33;CU, n = 22; CP, n = 24; C, n --- 17) were moved to a recording room and housed there individually in 6liter glass tanks (30 X 15 X 15 cm) for 3 days. All conditions were similar to rearing. The fish were tested in five replicated blocks as described by Gerlai et al. (1990). The genotypes of the fish were u n k n o w n to the observer at the time of the behavioral recording and the recording sequence of the fish was randomized across genotypes. The fish were monitored singly for 5 min in each o f the following four experimental situations. The first was the familiar 6-liter home tank in which the fish had been for 3 days. The second situation was an open field, a 70 • 70 x 20-cm glass tank painted white o n all but o n e side. Paradise fish in this situation exhibit a rich repertoire of exploratory behaviors (Csfinyi and Gerlai, 1988). On the bottom of the tank a 10 • 10-cm grid pattern was painted to m e a s u r e l o c o m o t i o n scores. The third s i t u a t i o n was mildly aversive (Gerlai and Cs/myi, 1990): a small (20 x 20 x 20-cm) transparent glass tank unfamiliar to the fish. The fourth situation was more aversive to the fish (Gerlai and Csfinyi, 1990). It was identical to the third, except that a rotating disk with white and black markings was placed 15 cm above the tank during the recording session. We recorded the sequence of mutually exclusive behavioral elements with a microcomputer event recorder program (see Gerlai and Hogan, 1992). The detailed definitions of behavioral elements have been published elsewhere (Csfinyi et al., 1985a, 1985b). Briefly, we recorded the relative durations of the following elements: staccato, a series of quick starts and sudden stops during l o c o m o t i o n ; swim, fast locomotion with the use of the caudal fin, the pectoral fins beating normally; move, slow locomotion without the use of the caudal fin; escape, rapid to-and-fro movement with forceful

387

swimming perpendicular to a glass wall; creep, being propelled forward only by pectoral fin fanning, with all other fins closed and the pectoral fins beating very quickly; oblique, the body axis of the immobile animal being inclined at 20-40 ~ from the horizontal plane, with the dorsal, caudal and anal fins closed and the pectoral fins quickly fanning as in creep; erratic, an intense, extremely rapid, zigzag-like locomotion; float, floating immobile just beneath the surface while holding position by beating the pectoral fins; hang, identical to float element but at medium water depth; rest, identical to float but at the bottom of the tank; and freeze, remaining motionless, with only the opercula, and occasionally the eyes, moving. We also recorded the frequencies of the following elements: leap, a quick jump due to a forceful lateral slap by the caudal fin; air gulp, swimming to the surface and gulping air (paradise fish being anabantoid); and pick, picking at small pieces of food or other visible spots with the mouth. In the open field, in addition to the above behavioral elements, we recorded the latency (s) of fish to emerge from a pot which was placed in the center of the field, as well as locomotion s c o r e s at three areas of the field, the inner area (OF inner), within 10 cm from the painted walls of the tank (OF wall), and within 10 cm from the transparent glass wall (OF glass). A score was counted when the fish entered a new square o f the grid pattem painted on the bottom of the tank. Genetic and Statistical Analysis

The monovariate analysis of variance and covariance of a replicated full diallel cross (Hayman, 1954; Jinks and Hayman, 1953) has been modified (Crusio et al., 1984) and extended (Crusio, 1993) to allow a bivariate analysis. We applied the bivariate extension o f the method to calculate genetical and e n v i r o n m e n t a l correlation coefficients. Briefly, bivariate equivalents o f the monovariate estimates o f E (environmental variance), D (additive genetic variance), and H2 (genetic variance due to dominance effects) were calculated by replacing the monovariate variance components with corresponding covariance components. For example, instead o f Vr (the variances of the arrays), we calculated Wr.~y(the covariances of arrays for variables x and y). The bivariate estimates of E~y, D~, and H2~ were divided by the square roots of the appropriate variance components (e.g., E J % /

388

Gerlai and Crusio

Table I. The Behavioral Elements of Paradise Fish Measured in the Familiar Home Tank (Untransformed Means +- SD)" Genotype

U

UP

UC

PU

P

PC

CU

CP

C

Esc~e Swim Move Staccato Creep Pick Air gulp Flo~ Hang Rest Oblique Freeze

14.2+-14.6 6.3+-5.1 47.7-+15.1 0.1+-0.3 0.2-+0.9 1.3+-1.7 5.6+-2.5 5.2-+8.0 8.8+-6.7 7.1• 0.2-+0.6 9.1-+14.5

11.4+-19.5 4.7-+6.2 44.9+-19.7 2.4+-8.5 0.1-+0.2 2.1+-3.0 3,7+-2.8 2.4-+4.0 13.8+-11.9 7.7-+7.3 0.5-+2.4 11.2+-18.3

20.2-+26.3 4.5-+7.5 23.8+-19.2 1.8-+4.1 2.0-+6.1 0.8• 2.8-+2.8 2.7• 9.1-+12.7 8.2-+9.2 0.8-+2.5 26.2-+30.2

13.2-+27.1 5.1-+5.9 35.9-+20.9 0.3-+1.0 0.3+-0.9 0.5-+0.8 3.9-+2.3 3.5+-5.0 12.7-+18.1 6.2+-8.0 0.0-+0.0 22.1+-31.3

3.6• 4.3-+6.3 40.7-+22.7 1.1+-2.0 1.8-+6.2 1.6-+2.4 3.1-+2.0 2.6-+5.3 14.9-+10.3 15.1-+22.3 0.0-+0.0 15.8-+24.6

3.2• 1.8+-3.0 7.8-+10.0 1.0+-2.4 1.8+-4.7 0.1-+0.2 0.8+-1.5 1.2-+3.5 8.5-+16.3 17.2-+18.8 0.3+-1.4 56.7+-33.4

11.2-+22.1 3.0-+3.5 30.5+-21.8 2.4-+3.0 1.4-+4.5 1.7-+2.6 4.3• 2.3-+5.1 14.3+-16.5 7.7• 3.5• 22.8-+27.7

4.7-+14.2 4.1+-5.9 22,2-+21.9 0,8-+2.5 0,9-+3.1 1.0+-2.3 1.5-+1.5 1.4+-2.5 13.5-+16.7 12.6• 0.4• 38.5-+41.1

13.9-+19.2 3.1+-3.5 32.9+-25.1 0.0-+0.0 1.2-+2.9 0.6+-1.3 3.1-+3.4 1.7-+3.5 10.2-+16.0 12.1• 1.2-+2.8 23.2-+35.6

The data on parental generations are in bold type. For the definitions of behavior elements and details of the experimental situations see Methods.

Table II. The Behavioral Elements of Paradise Fish Measured in the Large Open Field (Untransformed Means -+ SD) a I

i

Genotype

U

UP

UC

PU

P

PC

CU

Escape Swim Move Staccato Creep Erratic Pick Leap Air gulp Float Hang Rest Oblique Freeze Emerge OF inner OF wall OF glass

38.0___19.1 25.2-+11.7 28.1-+13.6 1.6+-3.1 2.3+-4.4 0.0-+ 0.0 0.7+-1.6 0.0 +- 0.0 12.2-+3.2 2.3+-1.7 0.7+-1.1 0.3-+1.1 0.3-+0.6 0.0-+ 0.0 4.0+-3.7 54+-85 48-+28 138-+51

32.3___17.2 27.0_+14.9 24.7-+12.5 3.8+-4.3 8.0-+10.3 0.0-+0.0 0.0+-0.0 0.2-+0.3 8.5-+2.4 2.2-+1.6 0.3+-0.6 0.1___0.3 0.3+-0.9 0.0-+ 0.0 10.1+-8.9 37+-17 77-+34 118+-54

31.7+__19.9 12.5-+8.9 23.0+-10.6 9.0-+8.8 19.4-+18.3 0.0 • 0.0 +-0.0 0.2-+0.2 6.5-+2.8 1.0-+1.5 0.1+-0.2 0.5-+1.0 1.4-+2.3 0.5-+2.8 23.4+-10.9 25-+12 35-+19 83+_48

18.0___13.1 17.0-+8.5 33.0+-10.5 10.6+-12.0 16.0-+11.6 0.1-4-0.1 0.6-+2.2 0.2-+0.4 7.5-+1.9 2.3-+3.5 0.2_+0.5 0.4+-1.2 1.4-+2.0 0.0-+ 0.0 13.4-+6.3 37-+17 60+-23 82_+48

13.2--+14.8 20.0_+10.7 34.9+-17.6 7.6___12.3 19.3-+21.0 0.1 -+ 0.1 0.5-+1.5 0.3+-0.4 6.5-+2.6 1.8+-3.3 0.5+_1.1 0.3+-0.8 1.2-+1.5 0.4-+1.2 17.4_+8.6 40-+16 63+-26 70-+48

13.0--+13.7 6.8-+6.2 11.1-+14.2 27,1-+16.1 30.5-+16.7 0.1 +-0.1 0.1-+0.3 0.1+-0.2 3.9+-2.0 1.4__2.5 0.2-+0.8 0.2+-0.6 6.7-+9.2 2.2-+9.3 38.3-+34.6 27-+9 26__18 49+-38

26.0-+14.7 13.9+__7.2 31.1-+12.9 8.7--+7.9 15.3+_11.8 0.0-+ 0.0 0.1-+0.2 0.2-+0.2 10.6-+3.5 1.4+-2.0 0.4-+0.7 0.4-+0.7 1.0___1.5 0.6-+1.7 20.5-+10.0 30+__10 36+-19 67-+21

CP

C

12.1• 6.6-+9.8 14.7----_12.7 8.1+--8.4 15.8-+13.1 24.9-+17.6 25.4-+15.2 5.9-+8.3 22.0-+16.8 38.0+-20.7 0.0 +-0.0 0.0-+ 0.0 0.0-+ 0.0 0.4-+1.5 0.1+-0.2 0.1 -+ 0.1 4.8-+2.3 5.8+-2.0 1.8+-3.2 9.6+-16.2 0.2-+0.6 0.2+-0.8 1.5-+6.3 0.2-+0.7 2.8-+3.7 4.6+-7.5 2.9-+6.2 0.8-+2.4 38.5-+17.9 70.6+-54.6 30+_13 35_+17 38-+21 21-+15 58+-37 33+-17

i

a The data on parental generations are in bold type. For the definitions of behavior elements and details of the experimental situations see Methods.

[E~Ey]) t o o b t a i n t h e e n v i r o n m e n t a l (re), additive g e n e t i c ( r o ) , a n d d o m i n a n c e (rH) c o r r e l a t i o n c o e f f i c i e n t s , r e s p e c t i v e l y . S t a t i s t i c a l s i g n i f i c a n c e o f Exy, Dxy, a n d H 2 ~ w a s e v a l u a t e d b y c a l c u l a t i n g t h e a p propriate standard errors according to the bivariate extension (Crusio, 1993) of Hayman's (1954) variance--covariance analysis. The normality and variance homogeneity criteria of the parametric statistical analyses were satisfied by applying scale

transformations according to the procedures of the BMDP (BioMedical Data Processing) software. The diallel cross analysis requires further assumptions: no epistasis, no multiple allelism, and independent distribution of alleles among the parental lines. These assumptions were tested by the reg r e s s i o n a n a l y s i s o f t h e Vr:VrcF-p) g r a p h , w h i c h should have a slope of -1 when the above criteria are fulfilled.

Behavioral Organization in Paradise Fish

389

Table IlL The Behavioral Elements of Paradise Fish Measured in the Small Unfamiliar Tank (Untransformed Means • SD) a Genotype

U

UP

UC

PU

P

PC

CU

CP

C

Esc~e Swim Move Staccato Cre~ Pick Leg Air g u ~ Flo~ H~g Rest Oblique Freeze

38.7-+17.3 12.8-+8.2 42.84-17.7 0.5• 0.4-+1.0 0.8-+3.6 0.0• 12.1• 1.3-+1.5 1.0• 0.6-+0.9 0.3• 0.0•

64.0• 10.1-+7.2 17.6-+14.5 1.9-+4.2 2.7• 0.0• 0.0-+0.0 10.7• 1.3• 0.3• 0.7• 0,2• 0.1•

38.2• 4.6• 35,3-+16.6 9,34-ll.4 8.2• 0.2-+0.6 0.0-+0.0 6.2• 0.2• 0.3• 1.3-+1.9 1.3-+1.8 0.6•

41.6-+19.2 8.8• 26.8• 8.9• 10.0• 0.1• 0.1• 9.2-+2.4 0.6-+1.2 0.2-+0.4 1.1• 1.0• 0.3-+1.1

32.2• 14.44-10.4 34.9• 9.4• 5.7• 0.0• 0.0• 8.4-+1.8 0.9• 0.3• 1.0-+1,6 0.6• 0.2•

26.8• 4.5• 32.7-+17.9 15.9-+17.9 12.6• 0.1• 0.1-+0.3 4.2• 0.2• 0.2• 3.04-2.6 0.9• 4.5-+12.0

26.84-19.6 5.0• 47.7• 9.5• 6.2• 0.0• 0.1-+0.2 9.1• 0.6• 0.5• 1.2• 0.6• 1.0•

17.04-15.0 4.9• 30.9• 19.8-+18.8 16.5• 0.0-+0.0 0.1-+0.2 5.3-+2.9 0.3-+0.7 0.2-+0.7 2.0• 1.7-+3.5 6.42-+17

4.6-+6.2 1.34-3.6 31.24-23.4 0.8-+1.2 38.34-22.6 0.1-+0.5 0.1-+0.1 5.34-4.3 0.9-+2.3 1.1-+1.6 2.2-+4.1 1.14-2.0 18.2-+27.1

~ see Methods.

Table IV. The Behavioral Elements of Paradise Fish Measured in the Small Unfamiliar Tank with a Rotating Disk Above (Untransformed Means • SD) ~ i

Genotype Escape Swim Move Staccato Creep Erratic Leap Air gulp Float Hang Rest Oblique Freeze

U

UP

65,1--+17.3 52.1-+20.8 4,8--+4.4 4.9_+6.5 18.9_+9.7 22.0• 0.7-+1.3 3.2_+5.5 2.7-+5.7 4.0-+6.1 0.3+0.7 0.4+1.2 0.3• 0.4_+0.6 10.24-5.0 8.04-3.7 0.8• 2.3• 0.2-+0.5 0.4-+1.0 2.9_+3.4 2.7+2.1 1.3___1.4 2.8• 1.5__+3.6 4.0-+5.6

UC

PU

50.1• 2.0-+3.1 13.5_+10.0 9.9• 3.1_+6.6 0.4+0.7

P

33.0• 18.9+21.9 2.1__3.7 3.3-+4.6 17.9_+18.7 25.7+18.8 3.2___3.9 12.0__16.4 9.2--+15.8 9.7_+7.2 0.6_+1.3 0.9_+1.3 0.5++_0,6 0.4_+0.4 0.7-+0.4 5.5-+3,4 5.0-+3.8 5.8-+3.5 0.7-+0.9 0.8+1.4 1.5+2.1 0.1_+0,2 0.1-+0.2 0.0-+ 0.0 2.3-+2.8 3.1+3.1 5.8_+9.0 0.8• 1.9-+2.2 2.7• 12.8_+19.5 27.0_+36.6 18.5_+26.6

PC

CU

35.9• 3.8_+5.2 16.5• 15.6-+18.1 7.3-+10.7 0.5-+1.1 0.4-+0.5 4.6-+3.1 0.6-+1.0 0.24-0.5 3.2-+3.0 2.6-+3.7 11.0-+17.3

35.5-----22.8 3.1 -+4.3 17.24-11.7 13.0-+11.2 5.1_4_-4.0 0.8-+1.3 0.4_+0.6 7.7-+4.6 0.4-+1.0 0.1-+0.2 1.8_+1.9 3.3-+5.2 17.7_+22.0

CP

C

27.5-----24.0 8.6• 2.1+3.5 0.4--+1.3 14.3-+12.6 18.3• 17.5-+17.9 1.8-+3.5 8.5_+12.5 18.3-+19.1 0.6-+1.0 0.2-+0.4 0.5-+0.7 0.34-0.4 4.1 -+3.8 3.6-+3.7 0.3-+1.2 1.1-+2.4 0.1-+0.3 0.4___1.1 2.7-+3.0 1.2-+2.1 2.0-+3.1 6.0-+5.9 23.64-32.0 42.94-40.0

a The data on parental generations are in bold type. For the definitions of behavior elements and details of the experimental situations see Methods.

In order to impart a parsimonious and concise d e s c r i p t i o n o f t h e c o r r e l a t i o n m a t r i c e s , a n d to r e veal possible underlying background factors, the c o r r e l a t i o n m a t r i c e s w e r e s u b j e c t e d to p r i n c i p a l component analysis (PCA) followed by a HarrisKaiser orthoblique rotation (Ray, 1982). The crit e n o n f o r i n c l u s i o n o f p r i n c i p a l f a c t o r s w a s s e t at e i g e n v a l u e s e q u a l t o o r g r e a t e r t h a n 1. V a r i a b l e s with nonsignificant effects were excluded from the PCA. Since additive genetic, dominance, and environmental correlations contribute portions to the t o t a l p h e n o t y p i c c o r r e l a t i o n t h a t a r e p r o p o r t i o n a l to

the square roots of the heritability in the narrow s e n s e (h2), t h e p o r t i o n o f t h e h e r i t a b i l i t y d u e s o l e l y to d o m i n a n c e e f f e c t s (h 2 - h2), a n d t h e e n v i r o n m e n t a l i t y (e 2 = 1 - h 2) r e s p e c t i v e l y , v a r i a b l e s w e r e weighted by these quantities.

RESULTS T h e b e h a v i o r a l d a t a o b t a i n e d in t h e f o u r e n vironmental situations are shown in Tables I through IV. The results of the monovariate analyses have been presented and discussed before (Gerlai

390

Gerlai

and Crusio

Table V. Phenotypical Principal-Factor Loading Structure" I

Principal factor Hang ~ Move t Oblique 3

i

2

3

4

5

6

7

9

10

0.93 0.65 -0.45

Rest 4

0.73

Float 1

0.55

Oblique 2

0.47

Rest 3

0.40

0.57 --0.40

Float 4

0.88

Move 4

0.86

Move 3

0.92

Pick 3

-- 0.42

0.44

OF walV

--0.36

Erratic 4

0.86

Staccato ~

0.44

Mov~

0.58

0.60

0.51

Staccato 3

0.39

Freeze 3

--0.38

--0.37

0.37

Swim 1

0.89

Swim 4

0.86

Swim 3

0.69

Erratic 2

0.37

0.64

Freeze 4

-0.51 --0.35

0.35 0.83

Staccato 2

--0.50

Staccato a

-0.58

1.12

Air gulp 1 0.37

Freeze ~

-0.56 -0.62

Escape 2

1.07

OF glass 2

0.99

Escape 4

0.79

Air gulp z

0.72

Air gulp 4

0.69

Air gulp 3

0.53

Escape 3

0.51

Emerge z

--0.57

Creep 4

--0.57

Creep 3

--0.71

Creep 2

--0.79

Oblique 4

0.46

0.91

Leap 4

0.44

--0.53

--0.53

Float 3

0.93 -0.55

--0.37

0.66

Float 2

0.50

Pick 2

0.99

Rest 2

0.93

Freeze z Swim 2

13

0.40

Hang z

Leap 2

12

--0.75

Creep a

Rest ~

11

0.99

Oblique ~

Escape ~

8

i

0.42 0.50

0.72 0.61 --0.38

14

15

Behavioral Organization in Paradise Fish

391

T a b l e V. C o n t i n u e d . Principal factor OF i n n e r 2 Hang 3 Leap 3 Hang4 Pick s

1

2

3

4

5

6

0.44 -- 0.44

7

8

9

10

11

12

13

14

15

0.93 0.47 --0.50 0.90 0.70

a O n l y m a j o r loadings, i.e., l o a d i n g s greater t h a n 10.35[, are included. For definitions o f behavioral e l e m e n t s a n d details o f the calculations see M e t h o d s . Superscripts o f behavioral e l e m e n t s indicate the e x p e r i m e n t a l situation in w h i c h t h e y were m e a s u r e d . ~First situation: familiar h o m e tank. 2Second situation: large o p e n field. 3Third situation: Small u n f a m i l i a r tank. 4Fourth situation: s m a l l u n f a m i l i a r t a n k w i t h a rotating disk above.

et aL, 1990). Briefly, strong genetic effects were observed and the four situations applied elicited distinct behavioral responses that were partly characteristic of a particular environment. In the home tank (Table I) the behavior of the fish was dominated by slow swimming (move) or motionless state (floating, hanging, resting, freezing), behaviors that are typical o f territorial or habituated fish (Csfinyi and Gerlai, 1988; Csfinyi et aL, 1985b). In the open field (Table II) fish were more active and exhibited locomotory behavior (swim, move) and escape. Members of the fear cluster (Csfinyi and Gerlai, 1988; Csfinyi et aL, 1985b; Gervai and Csfinyi, 1985) including creep, staccato, and oblique, also appeared. In the small novel tank (Table Ill) fish exhibited decreased swimming but increased escape. The values of the fear cluster elements were still high compared to those measured in the small home tank. In the small tank with the rotating disk (Table IV) paradise fish exhibited increased escape but decreased overall locomotion (swim, move). The fear cluster elements were especially pronounced and freezing was also regularly observed. A bivariate analysis o f these data revealed numerous significant phenotypical, environmental and genetic correlations among several behavioral elements (matrices not shown). The slope of the Vr: Vrcr-p) graph deviated significantly from - 1 in 144 o f 1540 bivariate cases, which is only slightly more than expected based on chance alone. Combined with the fact that in the monovariate analysis, significant violation of assumptions was found for one variable only [staccato in the familiar tank (see Gerlai et al., 1990)], we conclude that the additive dominance model fits our data reasonably well and

that a partitioning o f the covariance is allowed. Thus, the phenotypical, environmental, additive genetic, and dominance correlation matrices were subjected to principal-component analyses. The principal factors retained in the four analyses were submitted to an orthoblique rotation, explained at least 83% o f the total variance in each analysis, and are summarized in Tables V through VIII. The solution found was, generally, nearly orthogonal. In the PCA of the phenotypical correlation matrix, interfactor correlations were all smaller than ]0.40 I except those between PF1 and PF8 (r = 0.46), PF8 and PF13 (r = -0.42), PF9 and PF10 (r = -0.53), and P F l l and PF13 (r = -0.44). In the case o f the PCA of the environmental correlation matrix these correlations were all smaller than [0.30[ except between PF1 and PF13 (r = --0.46). The two PFs obtained in the PCA of the additive genetic correlation matrix were practically orthogonal (r = -0.07), whereas those extracted from the dominance correlation matrix were nearly so (PF1 and PF2, r = --0.27; PF1 and PF3, r = 0.12; PF2 and PF3, r -- -0.50). We have classified the principal factors (PFs) according to whether they characterize one (I), two (II), or more (III) situations as follows: (I) At least 75% o f the behaviors with major loadings have been measured in the same experimental situation (same superscript); (II) at least 75% o f the behaviors with major loadings have been measured in the same two experimental situations and the proportions o f behaviors of either situation did not exceed 75%; and (III) principal factors with major loadings o f behaviors have been measured in more than two situations or with only one major loading.

392

Gerlai

and Crusio

12

13

T a b l e V I . E n v i r o n m e n t a l P r i n c i p a l - F a c t o r L o a d i n g Structure ~ Principal f acto r OF g l a s s 2 E s c a p ea Swimz Creep2 P i c k3 Move 3 Hang 3 O b l i q u e4 Creep4 Staccato 4 Staccato 3 E r r a t i c4 Air gulp2 Freeze2 Move 2 Rest2 Freeze 1 O F irmer2 Staccato 2 Staccato 1 Oblique~ Creep ~ Swim4 Float3 Leap4 Swim 1 Swim3 E r r a t i c2 Pick2 Air gulp3 Float4 Move4 Obliquez Rest4 Rest3 Air gulp4 Pick ~ Hang4 Oblique3 Move 1 Float2 Leap2 Hang 1 Escape ~ Freeze4 Float ~ Freeze3 Escape4 Leap 3 Hang 2 Air gulp 1 Rest 1

3 0.99 0.86 0.65 - -0.67

4

0.36 --0.46 0.74 0.69 0.53

5

6

7

8

9

10

11

14

--0.38

-- 0.40 0.38 0.88 0.62 0.61 0.43 0.41

--0.36 0.36

0.36

0.35 0.35

0.38

0.84 --0.69 0.52 0.50

- -0.56

0.36

--0.37

0.44 --0.37 0.38 0.92 --0.48

0.40

0.37

0.36 0.93 0.58 0.55

--0.39

--0. 37

0.97 --0.58 0.37

--0.38

0.35

--0.55

--0.38 0.84 --0.69 --0.44 --0.51 0.95 --0.50 0.90 0.80 -0.61

--0.40

-0.39 0.46

0.91 0.55 --0.49 0.88 0.78 0.51 0.37

-0.37 --0.36 -0,40 --0.35

0.51

--0.35

0.37

0.99 -0.50 0.61 --0.48 0.43 0.42 --0.41 0.39 -0.39 0.84 0.52 0.43 --0.77

II

O n l y m a j o r l o a d i n g s , i.e., l o a d i n g s g r e a t e r t h a n 10.35[, are i nc l ude d. F or de fi ni t i ons o f b e h a v i o r a l e l e m e n t s a n d d e t a i l s o f the c a l c u l a t i o n s , see M e t h o d s . S u p e r s c r i p t s o f b e h a v i o r a l e l e m e n t s i n d i c a t e the e x p e r i m e n t a l s i t u a t i o n in w h i c h t h e y w e r e m e a s u r e d . 1First situation: f a m i l i a r h o m e tank. 2Second situation: l a rge o p e n field. 3Third situation: s m a l l u n f a m i l i a r tank. 4Fourth situation: s m a l l u n f a m i l i a r t a n k w i t h a r o t a t i n g d i s k above.

Behavioral Organization in Paradise Fish

393

Table VII. Additive-Genetic Principal-Factor Loading

Table VIII. Dominance-Genetic Principal Factor Loading

Structure ~

Structure-

Principal factor Float3 Move ~ Air gulp I Air gulp4 OF glass2 Escape4 Air g u l p 3 Escape z Air gulp 2 Swim~ Swim4 Escape 3 Swim3 Freeze3 Staccato2 Creep3 Creep4 Creep2 Emerge2 Freeze4 Obliquea Float4 Move2 Staccato3 Staccato4 OF wallz Oblique ~ Escape ~

1 1.05 1.03 1.02 1.01 1.01 1.00 0.98 0.97 0.96 0.95 0.95 0.94 0.78 -0.83 -0.88 -0.96 -0.98 - 1.01 -- 1.01 -- 1.01 - 1.02

0.58 -0.65 0.65

2

-0.39 -0.40

0.62 -0.55 0.79 0.36

1.26 1.09 1.00 0.99 0.79 -0.83 -0.93

Only major loadings, i.e., loadings greater than 10.351, are included. For definitions of behavioral elements and details of the calculations, see Methods. Superscripts of behavioral elements indicate the experimental situation in which they were measured. ~First situation: familiar home tank. zSecond situation: large open field. 3Third situation: small unfamiliar tank. 4Fourth situation: small unfamiliar tank with a rotating disk above.

In the P C A o f the p h e n o t y p i c a l correlations ( T a b l e V ) 15 p r i n c i p a l f a c t o r s w e r e r e t a i n e d : 5 characterize one experimental situation-PF1, PF2, PF4, PF5, and PF13 have major loadings of behaviors m e a s u r e d in t h e first, first, f o u r th , third, a n d second situation, respectively. PF10, PF14, and PF15 characterize two situations, the third-fourth, t h e s e c o n d - t h i r d , a n d th e f i r s t - f o u r t h , r e s p e c t i v e l y . The other principal factors characterize more than two situations or have only one m a j o r loading. The environmental principal factor structure ( T a b l e V I ) a p p e a r s to b e s i m i l a r to t h e p h e n o t y p i c a l

Principal factor Escape 3 OF waif Swim3 Move2 Air gulp I Air gulp2 Move I Float3 Escape4 Freeze 1 Staccato2 Rest 3 Staccato l Move3 Staccato~ Staccato3 Float2 Creep3

1 0.96 0.88 -0.93

--0.66

2

0.39 0.93 0.56 1.19 0.95 0.87 0.87 0.72 --0.85 --0.87 - 0.88 - 1.06 0.40

-0.71 -0.36 -0.57 0.37

3

0.40

--0.41

1.17 0.89 0.80 0.59 --0.95 --0.98

a Only major loadings, i.e., loadings greater than 10.351, are included. For definitions of behavioral elements and details of the calculations, see Methods. Superscripts of behavioral elements indicate the experimental situation in which they were measured. 1First situation: familiar home tank. 2Second situation: large open field. 3Third situation: small unfamiliar tank. 4Fourth situation: small unfamiliar tank with a rotating disk above.

one. H o w e v e r , the p r o p o r t i o n o f p r i n c i p a l f a c t o r s c h a r a c t e r i z i n g o n e o r t w o s i t u a t i o n s is s o m e w h a t h i g h e r (see a l s o Fig. 1): P F 1 , P F 4 , P F 6 , a n d P F 1 1 all s h o w m a j o r l o a d i n g s o f b e h a v i o r s m e a s u r e d in o n e s i n g l e s i t u a t i o n (the s e c o n d , s e c o n d , first, a n d f o u r t h situation, r e s p e c t i v e l y ) , w h e r e a s P F 2 , P F 3 , P F 5 , P F 7 , a n d P F 9 c h a r a c t e r i z e t w o s i t u a t i o n s (the second-third, third-fourth, second-third, thirdfourth, a n d f i r s t - t h i r d , r e s p e c t i v e l y ) . The additive genetic (Table VII) and domin a n c e g e n e t i c ( T a b l e V I I I ) p r i n c i p a l - f a c t o r struct u r es are m a r k e d l y d i f f e r e n t . T h e n u m b e r o f e x tracted PFs was small: T w o factors were extracted for the additive genetic and three for the d o m i n a n c e correlations. O n l y one genetic principal factor chara c t e r i z e d t w o s i t u a t i o n s (PF1 in T a b l e V I I I ) . A l l other factors had loadings o f behaviors o f more t h a n t w o e x p e r i m e n t a l si t u at i o n s.

DISCUSSION T h e b i v a r i a t e e x t e n s i o n ( C r u s i o , 1993) o f H a y m a n and Jinks' v a r i a n c e - c o v a r i a n c e analysis o f

394

Gerlai and Crusio

Phenotypical Principal Factor Structure

Environmental Principal Factor Structure

Genetic Principal Factor Structures

[]

Principal Factors characterizing 1 situation

[]

Principal Factors characterizing 2 situations

9 Principal Factors characterzing more than 2 situations Fig. 1. Proportion of principal components characterizing one, two, or more situations in the phenotypical, environmental, and (additive and dominance) genetic principal-component matrices. Note that the proportion of principal components characterizing multiple situations is smaller in the environmental principal-component matrix (Table III) and larger in the genetic principal-component matrices (Tables IV and V) compared to that of the phenotypical principal-component matrix (Table II). For definitions, see Results, the diallel cross made it possible for us to dissect the phenotypical correlations and estimate environmental, additive genetic, and dominance correlations. Since the n u m b e r o f pairwise correlations among several behavioral elements measured in different environmental situations is v e r y large (1540), we carried out principal-factor analyses to facilitate interpretation. It has been suggested (see, e.g., Fuller, 1979) that principal components extracted from a matrix o f phenotypical correlation between behavioral elements can be regarded as real traits in the sense that they represent a pattern o f organization typical o f the organism. W e have speculated (Csfinyi and Gerlai, 1988; Gerlai and Csfinyi, 1994) that, in paradise fish, such an organization m a y be due, at least in part, to higher neural systems controlling the coemergence o f the behavioral elements in response to specific environmental stimulus settings.

The phenotypical principal-factor pattern found in the present study is similar to previously obtained ones (Gerlai and Csfinyi, 1990, 1994), suggesting stable biological mechanisms underlying the observed correlations. W e have l a b e l e d these factors as behavioral states and the terminology used before is retained here. PF1 is "territoriality" in a familiar environment. It characterizes a slowly m o v i n g or motionless fish that does not exhibit exploratory or fear behaviors. PF2 is " f r i g h t e n e d s t a t e " in a familiar environment; it is characterized b y a typical cluster o f " f e a r " behaviors including staccato, oblique, and creep. PF3 is " p a s s i v i t y " across several situations. PF4 is " h a bituated state" in the situation with the predator model. It is characterized b y slow moving and floating passively. Note the abscence o f fear behaviors. PF5 is "habituated state" in small, transparent tank with no disk. PF6 is " f r i g h t e n e d state" across several situations. It is characterized b y the fear behavioral cluster. PF7 is " b a s i c activity" across several situations. It is characterized b y active swimming, i.e., l o c o m o t o r y behavior. The PF8 factor is most similar to that previously labeled " a c t i v e d e f e n s e " and is characterized b y escape and air-gulp and some behaviors associated with fear (staccato, freezing). The factor PF9 is also highly similar to " a c t i v e d e f e n s e . " PF10 has loadings o f behaviors belonging to the fear cluster, hence it was labeled " f r i g h t e n e d state." P F l l is "habituated state"; it is characterized by passive floating. For factor PF12, the only major loading is that o f pick. This factor is most probably associated with the level o f hunger. Factors PF13 and PF14 are similar to a factor previously labeled as " p a s sive d e f e n s e " in the open field, a behavioral state characterized b y high passivity (freezing or floating) and b y alarm response (leaping). PF15 is most similar to "habituated state" in novel situations. Although such labeling o f the phenotypical principal factors with descriptive names m a y facilitate visualization o f the behavioral response, the contribution o f environmental or genetic effects to this organization remains unknown. Since both the environment and the genotype could affect the organization o f complex behavioral responses and lead to the above described, phenotypically observable, correlation structure among behavioral elements, we investigated both components. Our results show a striking difference between the environmental and the genetic PCAs: Whereas the en-

Behavioral Organization in Paradise Fish

vironmental correlation structure is rather complex, the genetic one is relatively simple. The complex environmental correlation structure is characterized b y a relatively larger proportion o f principal factors specific to only one or two test situations (see also Fig. 1). W e postulate that there m a y be two fundamentally different sources o f environmental correlations and factors. First, small environmental variations intrinsic to a single test situation m a y induce environmental covariation between behavioral elements b y affecting one (or more) neural coordinating system(s). Such action m a y result in covariations between behavioral elements w i t h i n , but not between situations. In this way, one single neural coordinating system m a y give rise to multiple, statistically independent, situation specific factors. Second, during neural development epigenetic events m a y cause interindividual variations (Benno, 1990) which, not being o f genetic origin, are labeled " e n v i r o n m e n t a l " in quantitative genetic analyses. Environmental factors o f a PCA arising from such epigenetic events m a y be restricted to a particular stimulus combination or test situation, but not necessarily so. Epigenetic events m a y therefore lead to " e n v i r o n m e n t a l " covariations among behavioral elements both within and between situations. Currently, no m e t h o d exists to distinguish between the above possibilities. Genetical correlations m a y be due to pleiotropic gene effects or genetic linkage. Because the inbred strains used are highly unrelated, the existence o f a linkage disequilibrium is unlikely; yet given the low n u m b e r o f strains used, the possibility that accidental associations o f alleles within the inbred strains have given rise to some genetic correlations cannot be completely excluded. Pleiotropy is the more interesting possibility, because it implies that there exists a gene, or more probably a set o f genes, that simultaneously influences several traits. This means that at least part o f the physiological pathw a y leading from genotype to phenotype is shared for these traits. One m a y therefore conclude that a genetic factor is diagnostic for some form o f comm o n regulation o f the behavioral elements that load on it. In the present case, this suggests that the observed phenotypical multiformity o f paradise fish behavior has relatively simple genetic underpinnings. Here, simple should not be interpreted in terms o f n u m b e r o f genes, but in the sense that probably only a few (most likely not more than

395

five) genetically variable biochemical and/or developmental pathways, each influenced by an unspecified n u m b e r o f genes, underlie the observed behavioral variation. The complex and partly situation-specific nature o f the phenotypical correlation structure o f behavioral elements is then due to idiosyncratic influences on one or more o f these pathways, resulting in situation-specific environmental factors and, perhaps, to epigenetic events during neural development. In summary, our results demonstrate the following. First, both significant genetic and environmental correlations contribute to the observed phenotypical correlation structure o f the behavioral elements o f paradise fish. Second, the environmental correlations are often environment specific, while the genetic correlations are not. Finally, the observed complex phenotypical correlation structure is the result o f an underlying simple genetic correlation structure and a more c o m p l e x environmental correlation structure.

ACKNOWLEDGMENTS Thanks are due to Prof. J. A. H o g a n (Toronto) for his comments on the manuscript and to Prof. V. Csfinyi (Budapest), whose ideas initiated this study. R.G. was supported b y grant O T K A No. 2309 (Hungary) and b y M R C - C i b a - G e i g y (Canada); W.E.C., b y the CNRS (URA 1294), U F R Biom6dicale (Universit6 Paris V Ren~ Descartes), DRED, and the Fondation pour la Recherche M6dicale.

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Gerlai and Crusio

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