- Email: [email protected]
An asymmetric optomotor response in developing flounder larvae (Pseudopleuronectes americanus)
AN ASYMMETRIC OPTOMOTOR IN DEVELOPING FLOUNDER (PSEUDOPLEURONECTES
RESPONSE LARVAE
AMERICANW)
THO~IAS E. FINGER’ Department of Psychology and Brain Science, Massachusetts Institute of Technology. Cambridge. MA 02139. U.S.A. (Received
10 October
1975; in rrcisedform
18 December
1975)
Abstract-The optokinetic response of developing flounder larvae was examined for directional preference. Young symmetric larvae exhibit no such preference. However, older asymmetric larvae, in which the eyes have begun to migrate, show diminished responsiveness to stripes moving from their left-toright (clockwise as viewed from above). An explanation of this phenomenon may be neglect of input from the rapidly migrating eye. Key Words-flounder; (Pisces).
optokinetic
response: development;
ISTRODL-CTIOX
Flatfishes (flounders, soles and halibuts) are among the few bilaterally asymmetric animals. Adult flatfish have their two eyes on the same side of the body
and swim with the opposite lateral surface_downward. Yet, early in larval life, flatfish are entirely symmetric-one eye to each side of the body-and swim with their ventral aspect down as shown in Fig. 1. The duration of the larval period in winter flounder (Psewdoplrrcronecfes americanrts) at 8’C is approx 12 weeks, after which time the animal has an essentially adult, asymmetric form. For approx 5 weeks following hatching, the flounders are symmetric. After this time, the left eye begins a process of transposition or migration. Over a period of 3 weeks, the left eye migrates dorsally to cross the midline and ends up on the right side with its axis directed dorsally. During this same period, the right eye changes axis so as to be finally directed ventrally (Fig. 1). So, when the adult flounder lies on its side in the sand. the axes of both eyes are parallel to the ocean bottom. The metamorphic period for flatfish is thought to be one of great difficulty for the animal (Breder. 1955). Being visual feeders (Olla Samet and Studholme, 1972; Lawrence, personal communication), flounders cannot afford to completely ignore all visual inputs during the metamorphic period. Yet each day brings an altered relationship of eye axes to body axis. The animal keeps its visual axes nearly horizontal (personal observation) thereby slanting its body relative to vertical (see Fig. 1). Thus, it is not unlikely that a great degree of visuo-motor rearrangement is occuring during this time. This experiment is designed to test optomotor responses in larval flounders at different stages in development.
Pseudopkuronectes
omrricanus;
Telcostei
Marine Fisheries Facility located in Naragansett, R. I. Larvae from two clutches of eggs were used, the first having hatched 26 February and the second 4 May. At the time of testing. the larvae were approx 8 (9 mm) and 5 (7 mm) weeks old. For testing purposes, the animals were kept at MIT. in aerated sea water in a refrigerator (7’C). All animals
2
D
--__
-_
--a--_ \ d
V
3 --;*______
METHODS Subjects
Larval winter flounder (Pseudopfeuronecres americanus) were obtained from Dr. Jeffrey Latirence at the Sational ’ Author’s present address: Department of Anatomy, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
Fig. 1. Swimming attitude adopted by flatfish at various stages during development: (1) Young larva, pre-metamorphic; (2) Old larva, metamorphic; (3) Adult, post-metamorphic. D, dorsal: V. ventral: dotted lines indicate approximate optic axis for each eye. 941
942
T. E. FIXER
tested were at LM.LT.no longer than 6 days. During this period, mortality was below 15”;. ,After 2 weeks at M.I.T. approx 507; of the animals remained alive. The fish were treated as two groups according to age. The older larvae all had asymmetric eyes, i.e. stage 4b (Ryland, 1966) or older. The young larvae all had symmetric eyes, stage 3 or earlier.
Older animals would occasionally come to rest on the bottom of the fish chamber and not begin swimming within 3 set following onset of stimulus rotation. Usually, optokinetic nqsragmus could be observed during this period. In these cases, rotation was halted and that trial not counted. For each trial, a response index (RI) was calculated
according to the formula: RI = if’dl x 100 where/‘= score of the fish and d = stimulus drum rpm. For each subject. the mean RI value for rotation in each direction was calculated.
Prorrdurr
Initial tests were carried out in an optomotor device turned by hand but later tests were accomplished in a similar motor-driven device as described below. Both optomofor apparatus consisted of three compartments: fish chamber, stimulus container and cooling tray. The fish chamber, innermost of the three, was a crystallizing dish (25 mm deep x 70 mm dia) with an opaque white bottom. Cold (8°C) sea water was placed in the dish fo a depth of 20 mm. Surrounding this chamber, the striped stimulus field was placed in a glass stimulus container 100mm dia filled with cold sea water to the same level as in the fish chamber. As both fish and stimulus were submerged in sea water, no air-water interface existed between the two. The stimulus field consisted of alternating vertical blacklight stripes approx 9 mm wide on a plastic drum 82 mm dia. Thus, for a fish in the center of the container in the middle of the water column, the unrefracted stimulus field was approx 20” high for a 360’ circle around the animal. The stimulus container was surrounded by a 12.5 x 17.5 cm tray containing an ice bath which maintained the fish chamber temperature at 7-10°C.
RESLLTS All subjects exhibited optomotor responses during the period of drum rotation. Swimming patterns observed during this response were identical to those reported by Shaw and Sachs (1967) for Men& larvae. During periods when the drum was motionless. the Hounders exhibited seemingly random swimming patterns with no obvious preference for turning right or left. Younger larvae (numbered individuals) showed no consistent preference for stimuli rotating clockwise (C) or counter-clockwise (CC) although some individuals exhibited such preferences (see Fig. 2). In contrast, nine of ten older animals showed greater response to CC than to C stimuli. Young, symmetric larvae, when taken as a group show equally large RIs for clockwise (C) as well as counter-clockwise (CC) moving stimuli. However, some individuals within the group e.xhibit significant (P = 93; using a t-test) preference for stripes moving in one or the other direction (see Fig. 2). Exactly half the younger individuals show a preference for CC stimuli, the others preferring C stimuli. Older. asymmetric larvae show a marked and significant decrease in RI for clockwise (C) stimuli when compared to the younger larvae. Yet, response to CC rotation, as measured by RI. is not much different in older and younger larvae. Nearly all individuals within the older group exhibit a greater RI for the counter-clockwise movement. In only one individual
Test procedure
The fish was allowed 10-20 min to acclimate to the test chamber prior to each testing session. For each trial, the animal was observed for the 1 min of drum rotation. Between each trial. a 30 set rest period was permitted. The number of body rotations executed by the fish was counted during the test period. Orientation of an animal was determined by its sagittal plane. Turns by the fish in the same direction as the drum were counted as positive while turns opposite to the drum’s rotation were scored as negative. For example, a fish executing six turns in the same direction as the drum and one turn in the opposite direction, was assigned a score (F) of + 5.
mc
ImmaICC
IO
4 :
YOUNG
I
’I
OLD
TOTAL I
: : :
Fig. 2. IMean response index for all subjects and for pooled data from either all pre-metamorphal (YNG) or metamorphal (OLD) larvae. Solid bars for clockwise stimuli: broken bars for countertlockwise stimuli. Asterisk above bars indicates the difference in means is significant (P < 0.05) using the t-test. Circled asterisk shows only case in which clockwise movement elicited significantly more response than counter-clockwise movement.
Asymmetric optomotot response in P. americanus
Table 1
Mean RI
YC
YCC
oc
56.9
62.7
36’
occ
58.2
* :-test P < 0.05
out of the 10 older fish was there a greater C than CC response index. Table 1 shows a comparison of the mean RI values for the pooled data from each of the following groups: young larvae. clockwise stimuli (YC): young larvae. counter-clockwise stimuli (YCC): old larvae, clochx-ise stimuli (OC); old larvae, counter-clockwise stimuli (OCC). Using the r-test, it was found that OC is significantly (P = 95?,) lower than any of the other groups (YC. YCC. OCC) or the pooled data from these groups: and that these other groups do not differ significan,tly from each other or the pooled data of the remammg groups. In summary, an asymmetrical optokinetic response to differently directed moving fields was found only in the older, asymmetrical larvae. The asymmetry is in the form of a reduced response to stripes moving clockwise (left to right) relative to the animal. DISCUSSION The optomotor response of larval winter flounder was studied using two age groups: 5 weeks (pre-metamorphai) and 8 weeks (metamorphal). In the younger group, the fish swam upright and showed no asymmetry in eye position. Nearly all of the older group swam with the dorso-ventral axis of their body at some acute angle to horizontal (Fig. 1) and had asymmetrically placed eyes. The younger group showed no consistent preferred direction of movement for maximal responses to the stimulus. In contrast, nine of the 10 older larvae exhibited greater responses to counterclockwise (left-toright) than to clockwise (right-to-left) moving stimuli. Adult flatfish have been shown (Harden-Jones, 1963) not to exhibit an optomotor response in which the animal swims about the tank. Rather, adults do give binocular nystagmus in response to a moving striped field. Some of the older larvae would occasionally exhibit similar behavior by remaining on the bottom of the fish chamber after the onset of stimulus movement. Shaw and Sachs (1967) tested optomotor responses in developing Menidia menidia. Their data indicate that these fish show a higher response level, as measured by RI (see procedures) than do any of the flounder larvae. This may represent an actual species difference or may merely reflect slight differences in the testing apparatus. Shaw and Sachs do not report a directional preference for Me&in but no analysis for this phenomenon is reported either. A number of possible simple explanations can be offered to explain the asymmetry of response in older flounder larvae. Among them are:
(1) The asymmetric flounder, being canted to one side, finds it easier to swim to the left (CC) than to the right (C). (2) The asymmetric animal ignores all information coming to the left eye and responds primarily to stimuli impinging upon the right eye. In addition, the animal responds better to stimuli moving nasalward (CC) than to stimuli moving temporally (C). The first explanation seems not to be the case as the hounder larvae show no preference in turning right or left during the periods of free swimming prior to testing. However, this was not carefully examined and warrants more precise study. The second explanation has some supporting evidence for at least one of the assumptions. Easter (1972) demonstrated that in goldfish, stimuli moving from posterior to anterior (nasalward) are more effective in eliciting optokinetic nystagmus than stimuli moving in the opposite direction. In late metamorphal stage larval flounder (stage 4c+). the same appears to hold true (Finger. unpub. obr.1. No such greater effectiveness of rostralward-moving stimuli has been shown for the optomotor response. However, if it is assumed that such a condition exists. it is possible to account for the asymmetric response found in this study. If the flounder discounts visual inputs to the rapidly migrating left eye. then one would expect rostralward-moving stripes (CC for the right eye) to elicit a greater optomotor response than caudalward-moving stripes (C for the right eye). This hypothesis can be easily tested bv occluding or ablating the left eye of pre-metamorphic larvae. These young larvae should then show decreased optomotor response to stimuli moving clock&se. If the left eye of older larvae is occluded. little effect on the optomotor response should be evident when compared to larvae of the same age whose eye is not occluded. Acknow[engemenrs-The author is grateful to Dr. Jeffrey Lawrence for supplying the larval flounders. In addition. thanks are due to Drs. R. Held and W. Richards for useful comments and criticism of the manuscript. This work was supported by The Grant Foundation and the MIT. Department of Psychology.
REFERENCES
Breder C. M. Jr. (1955) Special features of visual reduction in flatfishes. Zoologica-40, 91-98. Easter S. (1972) Pursuit eve movements in goldfish (Carussius auratus): Won Re;. 12. 673-688. Harden-Jones F. R. (1963) The reaction of fish to moving backgrounds. 1. exp. Biol. 40, 437-446. Olla B.. Samet C. E. and Studholme A. L. (1971) Activity and feeding behavior of the summer Rounder (Paralichth,vs denmtus).
U.S. nat. mar. Fish
Ser.
Fish
Bull.
70, 1127-1136. Ryland J. S. (1966) Observations on the de\-elopment of larvae of the plaice, Plectronectes plaressa L_ in aquaria. 1. Cons. perm. inr. Explor. Mer 30, 177-195. Shaw E. and Sachs B. J. (1967) Developmenr of the optomotor response in the schooling fish Media menidLI J. camp. phys. Psychol. 63, 355-358.
RESPONSE LARVAE
AMERICANW)
THO~IAS E. FINGER’ Department of Psychology and Brain Science, Massachusetts Institute of Technology. Cambridge. MA 02139. U.S.A. (Received
10 October
1975; in rrcisedform
18 December
1975)
Abstract-The optokinetic response of developing flounder larvae was examined for directional preference. Young symmetric larvae exhibit no such preference. However, older asymmetric larvae, in which the eyes have begun to migrate, show diminished responsiveness to stripes moving from their left-toright (clockwise as viewed from above). An explanation of this phenomenon may be neglect of input from the rapidly migrating eye. Key Words-flounder; (Pisces).
optokinetic
response: development;
ISTRODL-CTIOX
Flatfishes (flounders, soles and halibuts) are among the few bilaterally asymmetric animals. Adult flatfish have their two eyes on the same side of the body
and swim with the opposite lateral surface_downward. Yet, early in larval life, flatfish are entirely symmetric-one eye to each side of the body-and swim with their ventral aspect down as shown in Fig. 1. The duration of the larval period in winter flounder (Psewdoplrrcronecfes americanrts) at 8’C is approx 12 weeks, after which time the animal has an essentially adult, asymmetric form. For approx 5 weeks following hatching, the flounders are symmetric. After this time, the left eye begins a process of transposition or migration. Over a period of 3 weeks, the left eye migrates dorsally to cross the midline and ends up on the right side with its axis directed dorsally. During this same period, the right eye changes axis so as to be finally directed ventrally (Fig. 1). So, when the adult flounder lies on its side in the sand. the axes of both eyes are parallel to the ocean bottom. The metamorphic period for flatfish is thought to be one of great difficulty for the animal (Breder. 1955). Being visual feeders (Olla Samet and Studholme, 1972; Lawrence, personal communication), flounders cannot afford to completely ignore all visual inputs during the metamorphic period. Yet each day brings an altered relationship of eye axes to body axis. The animal keeps its visual axes nearly horizontal (personal observation) thereby slanting its body relative to vertical (see Fig. 1). Thus, it is not unlikely that a great degree of visuo-motor rearrangement is occuring during this time. This experiment is designed to test optomotor responses in larval flounders at different stages in development.
Pseudopkuronectes
omrricanus;
Telcostei
Marine Fisheries Facility located in Naragansett, R. I. Larvae from two clutches of eggs were used, the first having hatched 26 February and the second 4 May. At the time of testing. the larvae were approx 8 (9 mm) and 5 (7 mm) weeks old. For testing purposes, the animals were kept at MIT. in aerated sea water in a refrigerator (7’C). All animals
2
D
--__
-_
--a--_ \ d
V
3 --;*______
METHODS Subjects
Larval winter flounder (Pseudopfeuronecres americanus) were obtained from Dr. Jeffrey Latirence at the Sational ’ Author’s present address: Department of Anatomy, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.
Fig. 1. Swimming attitude adopted by flatfish at various stages during development: (1) Young larva, pre-metamorphic; (2) Old larva, metamorphic; (3) Adult, post-metamorphic. D, dorsal: V. ventral: dotted lines indicate approximate optic axis for each eye. 941
942
T. E. FIXER
tested were at LM.LT.no longer than 6 days. During this period, mortality was below 15”;. ,After 2 weeks at M.I.T. approx 507; of the animals remained alive. The fish were treated as two groups according to age. The older larvae all had asymmetric eyes, i.e. stage 4b (Ryland, 1966) or older. The young larvae all had symmetric eyes, stage 3 or earlier.
Older animals would occasionally come to rest on the bottom of the fish chamber and not begin swimming within 3 set following onset of stimulus rotation. Usually, optokinetic nqsragmus could be observed during this period. In these cases, rotation was halted and that trial not counted. For each trial, a response index (RI) was calculated
according to the formula: RI = if’dl x 100 where/‘= score of the fish and d = stimulus drum rpm. For each subject. the mean RI value for rotation in each direction was calculated.
Prorrdurr
Initial tests were carried out in an optomotor device turned by hand but later tests were accomplished in a similar motor-driven device as described below. Both optomofor apparatus consisted of three compartments: fish chamber, stimulus container and cooling tray. The fish chamber, innermost of the three, was a crystallizing dish (25 mm deep x 70 mm dia) with an opaque white bottom. Cold (8°C) sea water was placed in the dish fo a depth of 20 mm. Surrounding this chamber, the striped stimulus field was placed in a glass stimulus container 100mm dia filled with cold sea water to the same level as in the fish chamber. As both fish and stimulus were submerged in sea water, no air-water interface existed between the two. The stimulus field consisted of alternating vertical blacklight stripes approx 9 mm wide on a plastic drum 82 mm dia. Thus, for a fish in the center of the container in the middle of the water column, the unrefracted stimulus field was approx 20” high for a 360’ circle around the animal. The stimulus container was surrounded by a 12.5 x 17.5 cm tray containing an ice bath which maintained the fish chamber temperature at 7-10°C.
RESLLTS All subjects exhibited optomotor responses during the period of drum rotation. Swimming patterns observed during this response were identical to those reported by Shaw and Sachs (1967) for Men& larvae. During periods when the drum was motionless. the Hounders exhibited seemingly random swimming patterns with no obvious preference for turning right or left. Younger larvae (numbered individuals) showed no consistent preference for stimuli rotating clockwise (C) or counter-clockwise (CC) although some individuals exhibited such preferences (see Fig. 2). In contrast, nine of ten older animals showed greater response to CC than to C stimuli. Young, symmetric larvae, when taken as a group show equally large RIs for clockwise (C) as well as counter-clockwise (CC) moving stimuli. However, some individuals within the group e.xhibit significant (P = 93; using a t-test) preference for stripes moving in one or the other direction (see Fig. 2). Exactly half the younger individuals show a preference for CC stimuli, the others preferring C stimuli. Older. asymmetric larvae show a marked and significant decrease in RI for clockwise (C) stimuli when compared to the younger larvae. Yet, response to CC rotation, as measured by RI. is not much different in older and younger larvae. Nearly all individuals within the older group exhibit a greater RI for the counter-clockwise movement. In only one individual
Test procedure
The fish was allowed 10-20 min to acclimate to the test chamber prior to each testing session. For each trial, the animal was observed for the 1 min of drum rotation. Between each trial. a 30 set rest period was permitted. The number of body rotations executed by the fish was counted during the test period. Orientation of an animal was determined by its sagittal plane. Turns by the fish in the same direction as the drum were counted as positive while turns opposite to the drum’s rotation were scored as negative. For example, a fish executing six turns in the same direction as the drum and one turn in the opposite direction, was assigned a score (F) of + 5.
mc
ImmaICC
IO
4 :
YOUNG
I
’I
OLD
TOTAL I
: : :
Fig. 2. IMean response index for all subjects and for pooled data from either all pre-metamorphal (YNG) or metamorphal (OLD) larvae. Solid bars for clockwise stimuli: broken bars for countertlockwise stimuli. Asterisk above bars indicates the difference in means is significant (P < 0.05) using the t-test. Circled asterisk shows only case in which clockwise movement elicited significantly more response than counter-clockwise movement.
Asymmetric optomotot response in P. americanus
Table 1
Mean RI
YC
YCC
oc
56.9
62.7
36’
occ
58.2
* :-test P < 0.05
out of the 10 older fish was there a greater C than CC response index. Table 1 shows a comparison of the mean RI values for the pooled data from each of the following groups: young larvae. clockwise stimuli (YC): young larvae. counter-clockwise stimuli (YCC): old larvae, clochx-ise stimuli (OC); old larvae, counter-clockwise stimuli (OCC). Using the r-test, it was found that OC is significantly (P = 95?,) lower than any of the other groups (YC. YCC. OCC) or the pooled data from these groups: and that these other groups do not differ significan,tly from each other or the pooled data of the remammg groups. In summary, an asymmetrical optokinetic response to differently directed moving fields was found only in the older, asymmetrical larvae. The asymmetry is in the form of a reduced response to stripes moving clockwise (left to right) relative to the animal. DISCUSSION The optomotor response of larval winter flounder was studied using two age groups: 5 weeks (pre-metamorphai) and 8 weeks (metamorphal). In the younger group, the fish swam upright and showed no asymmetry in eye position. Nearly all of the older group swam with the dorso-ventral axis of their body at some acute angle to horizontal (Fig. 1) and had asymmetrically placed eyes. The younger group showed no consistent preferred direction of movement for maximal responses to the stimulus. In contrast, nine of the 10 older larvae exhibited greater responses to counterclockwise (left-toright) than to clockwise (right-to-left) moving stimuli. Adult flatfish have been shown (Harden-Jones, 1963) not to exhibit an optomotor response in which the animal swims about the tank. Rather, adults do give binocular nystagmus in response to a moving striped field. Some of the older larvae would occasionally exhibit similar behavior by remaining on the bottom of the fish chamber after the onset of stimulus movement. Shaw and Sachs (1967) tested optomotor responses in developing Menidia menidia. Their data indicate that these fish show a higher response level, as measured by RI (see procedures) than do any of the flounder larvae. This may represent an actual species difference or may merely reflect slight differences in the testing apparatus. Shaw and Sachs do not report a directional preference for Me&in but no analysis for this phenomenon is reported either. A number of possible simple explanations can be offered to explain the asymmetry of response in older flounder larvae. Among them are:
(1) The asymmetric flounder, being canted to one side, finds it easier to swim to the left (CC) than to the right (C). (2) The asymmetric animal ignores all information coming to the left eye and responds primarily to stimuli impinging upon the right eye. In addition, the animal responds better to stimuli moving nasalward (CC) than to stimuli moving temporally (C). The first explanation seems not to be the case as the hounder larvae show no preference in turning right or left during the periods of free swimming prior to testing. However, this was not carefully examined and warrants more precise study. The second explanation has some supporting evidence for at least one of the assumptions. Easter (1972) demonstrated that in goldfish, stimuli moving from posterior to anterior (nasalward) are more effective in eliciting optokinetic nystagmus than stimuli moving in the opposite direction. In late metamorphal stage larval flounder (stage 4c+). the same appears to hold true (Finger. unpub. obr.1. No such greater effectiveness of rostralward-moving stimuli has been shown for the optomotor response. However, if it is assumed that such a condition exists. it is possible to account for the asymmetric response found in this study. If the flounder discounts visual inputs to the rapidly migrating left eye. then one would expect rostralward-moving stripes (CC for the right eye) to elicit a greater optomotor response than caudalward-moving stripes (C for the right eye). This hypothesis can be easily tested bv occluding or ablating the left eye of pre-metamorphic larvae. These young larvae should then show decreased optomotor response to stimuli moving clock&se. If the left eye of older larvae is occluded. little effect on the optomotor response should be evident when compared to larvae of the same age whose eye is not occluded. Acknow[engemenrs-The author is grateful to Dr. Jeffrey Lawrence for supplying the larval flounders. In addition. thanks are due to Drs. R. Held and W. Richards for useful comments and criticism of the manuscript. This work was supported by The Grant Foundation and the MIT. Department of Psychology.
REFERENCES
Breder C. M. Jr. (1955) Special features of visual reduction in flatfishes. Zoologica-40, 91-98. Easter S. (1972) Pursuit eve movements in goldfish (Carussius auratus): Won Re;. 12. 673-688. Harden-Jones F. R. (1963) The reaction of fish to moving backgrounds. 1. exp. Biol. 40, 437-446. Olla B.. Samet C. E. and Studholme A. L. (1971) Activity and feeding behavior of the summer Rounder (Paralichth,vs denmtus).
U.S. nat. mar. Fish
Ser.
Fish
Bull.
70, 1127-1136. Ryland J. S. (1966) Observations on the de\-elopment of larvae of the plaice, Plectronectes plaressa L_ in aquaria. 1. Cons. perm. inr. Explor. Mer 30, 177-195. Shaw E. and Sachs B. J. (1967) Developmenr of the optomotor response in the schooling fish Media menidLI J. camp. phys. Psychol. 63, 355-358.