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THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

GARY N. CALKINS,

E. G. CONKLIN, Princeton University E. N. HARVEY, Princeton University SELIG HECHT, Columbia University LEIGH HOADLEY, Harvard University M. H. JACOBS, University of Pennsylvania H. S. JENNINGS, Johns Hopkins University E. E. JUST, Howard University

Columbia University

FRANK R. LILLIE, University of Chicago CARL R. MOORE, University of Chicago GEORGE T. MOORE, Missouri Botanical Garden T. H. MORGAN, California Institute of Technology G. H. PARKER, Harvard University W. M. WHEELER, Harvard University EDMUND B. WILSON, Columbia University

ALFRED C. REDFIELD, Harvard University Managing Editor

VOLUME LXX

FEBRUARY TO JUNE, 1936

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE & LEMON STS.

LANCASTER, PA.

LANCASTER PKKSS, INC., LANCASTKR, PA.

CONTENTS

No. 1. FEBRUARY, 1936

PAGE

PARKER, G. H.

Integumentary Color Changes in the Newly-born Dogfish, Mustelus canis 1

BROWN, F. A.

Light Intensity and Melanophore Response in the Minnow, Ericymba buccata Cope 8

GRAFFLIN, ALLAN L., AND R. G. GOULD, JR.

Renal Function in Marine Teleosts. II. The nitrogenous constituents of the urine of sculpin and flounder, with par- ticular reference to trimethylamine oxide 16

HENSHAW, P. S., AND D. S. FRANCIS

The Effect of X-Rays on Cleavage in Arbacia Eggs: Evidence

of Nuclear Control of Division Rate 28

SANDSTROM, CARL J.

Reciprocal Chorio-allantoic Transplants of Embryonic Duck and Chick Kidney Transplants 36

POMERAT, CHARLES MARC

Experimental Studies on the Nuptial Pads of Male Triturus viridescens 50

STABLER, ROBERT M., AND TZE-TUAN CHEN

Observations on an Endamoeba Parasitizing Opalinid Ciliates 56

CHEN, TZE-TUAN, AND ROBERT M. STABLER

Further Studies on the Endamoebae Parasitizing Opalinid Ciliates 72

COHEN, ARTHUR, AND N. J. BERRILL

The Early Development of Ascidian Eggs 78

FRY, HENRY J.

Studies of the Mitotic Figure. V. The time schedule of mitotic changes in developing Arbacia eggs 89

WHITAKER, D. M.

The Effect of White Light upon the Rate of Development of the Rhizoid Protuberance and the First Cell Division in Fucus furcatus. 100

iv CONTEXTS

PAGE

BARNES, T. CUNLIFFE

Experiments on Ligia in Bermuda. IV. The effects of heavy water and temperature 109

FISH, CHARLES J.

The Biology of Calanus finmarchicus in the Gulf of Maine and Bay of Fundy. . . 118

STUMP, A. B.

The Influence of Test Materials on Reproduction in Pontigu- lasia vas (Leidy) Schouteden 142

RENN, CHARLES E.

The Wasting Disease of Zostera marina 148

No. 2. APRIL, 1936

KLEINHOLZ, L. H.

Crustacean Eye-stalk Hormone and Retinal Pigment Migra- tion 159

WHEELER, WILLIAM MORTON

Binary Anterior Ocelli in Ants 185

FISH, CHARLES J.

The Biology of Pseudocalanus minutus in the (iulf of Maine and Bay of Fundy . 193

WELSH, JOHN H.

Diurnal Movements of the Eye Pigments of Anchistioides . . . 217

GRAFFLIN, ALLAN L.

Renal Function in Marine Teleosts. III. The excretion of urea 228

SPARROW, F. K., JR.

Biological Observations on the Marine Fungi of Woods Hole Waters 236

LACKEY, JAMES B.

Occurrence and Distribution of the Marine Protozoan Species

in the Woods Hole Area. 264

GUTTMAN, S. A.

Effect of Ultraviolet Radiation on the Heart of Limulus poly- phemus 279

WKI.SH, MARTHA F.

Oxygen Production by Zooxanthellae in a Bermudan Turbel- larian 282

LAWSON, CHESTER A.

A Chromosome Study of the Aphid Macrosiphum solanifolii. . 288

FULLER, JOHN L., AND GEORGE L. CLARKK

Further Experiments on the Feeding of Calanus finmarchicus. 308

CONTENTS v

PAGE

TERROUX, KATHLEEN GODWIN

The Buffering Powers of Natural and Dialysed Helix pomatia Serum 321

GRAVE, B. H., AND JAY SMITH

Sex Inversion in Teredo navalis and its Relation to Sex Ratios 332

HlRAIWA, YOSHI KUNI, AND TOSHIJIRO KAWAMURA

Relation between Maturation Division and Cleavage in Arti- ficially Activated Eggs of Urechis unicinctus (von Drasche) . . 344

No. 3. JUNE, 1936 FAURE-FREMIET, E.

The Folliculinidae (Infusoria heterotricha) of the Breton

Coast 353

CLARK, JEAN M.

An Experimental Study of Polyspermy 361

KALISS, NATHAN, AND MARC A. GRAUBARD

The Effect of Temperature on Oviposition in Drosophila

melanogaster 385

PUCKETT, WILLIAM O.

The Effects of X-Radiation on the Regeneration of the Hy-

droid, Pennaria tiarella 392

CHURNEY, LEON

The Quantitative Determination of Mitotic Elongation 400

MAST, S. O., AND BRAINARD HAWK

Response to Light in Peranema trichophorum 408

HOPKINS, A. E.

Pulsation of Blood Vessels in Oysters, Ostrea lurida and O.

gigas 413

HETHERINGTON, ALFORD

The Precise Control of Growth in a Pure Culture of a Ciliate,

Glaucoma pyriformis 426

SAYLES, LEONARD P.

Regeneration in the Polychaete Clymenella torquata. III.

Effect of level of cut on type of new structures in anterior

regeneration 441

COONFIELD, B. R.

Apical Dominance and Polarity in Mnemiopsis Leidyi, Agassiz 460 WAKSMAN, SELMAN A., AND CHARLES E. RENN

Decomposition of Organic Matter in Sea Water by Bacteria. . 472 SCHRADER, FRANZ

The Kinetochore or Spindle Fibre Locus in Amphiuma tri-

dactylus 484

Vol. LXX, No. 1 February, 1936

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

INTEGUMENTARY COLOR CHANGES IN THE NEWLY-BORN DOGFISH, MUSTELUS CANIS

G. H. PARKER

(From the Biological Laboratories, Harvard University)

INTRODUCTION

It is well-known that the integumentary melanophores of the lower vertebrates are functionally active at a very early stage. These color- cells become responsive at about the time young fishes hatch from their eggs. This is true of Fundulus, according to Bancroft (1912), Spaeth (1913), and Gilson (1926) and of Coregonus and of two species of Salmo according to Becher (1929). At this stage the melanophores begin to disperse or concentrate their pigment, thus giving to the young fish a dark or a light tint. Although the beginnings of larval life may be said thus to mark the period of the first color changes, Becher noted that the embryos of fishes on which he worked when artificially removed from their egg-shells would often show color changes. This demonstrated that melanophore activity is possible at least under experimental conditions earlier than the time of hatching. Such a view was also taken by Duspiva (1931), who worked upon two fishes, Salmo salvelinus and Percafluviatilis. In Perca the first integu- mentary melanin appears five days after the eggs have been fertilized. Duspiva saw melanophore responses three days later or about eight days after fertilization. He believed, however, that responses may have occurred still earlier. As the young larvse of Perca hatch from the eggs about ten days after fertilization, the first melanophore responses in this fish must occur in what is obviously its embryonic period. From all his observations Duspiva was led to conclude that melanophores probably become active as soon as their processes are developed and their melanin formed. Such a view, which appears to be fairly well supported, places the initiation of melanophore activity in fishes at a very early stage, not later than about the time of hatching and probably in some species somewhat earlier.

The first responses of melanophores in lower vertebrates were shown by Babak (1910) to be quite unlike those of later life. In the

1

2 G. H. PARKER

very young larva? of the Mexican axolotl about 1.5 cm. long, when the first color changes had begun, the following conditions were found by Babak. In complete darkness the young animals were pale and in bright light they were dark. \Yhen they had attained a length of about 5 cm. their reactions in these respects were almost reversed. At this later stage they were in complete darkness dark and in bright light either pale or dark depending upon their surroundings. This second stage of their melanophore responses persisted until maturity when they became dark and remained permanently so, having lost their capacity to change. The early larval condition may be called the primary phase, the succeeding one the secondary phase. If a larva in the secondary phase is blinded, it was found by Babak to revert to the primary phase in that it would be pale in darkness and dark in bright light. A young larva in the primary phase when de- prived of its eyes remained unchanged and was responsive in the same way as it had been before the operation. From these tests Babak concluded that what has been called here the primary phase was dependent upon the direct stimulation of the larval melanophores by light or its absence and that the secondary phase resulted from their indirect stimulation through the eyes of the larva. These conclusions were supported in one way or another by Pernitzsch's work (1913) on axolotl, by Hooker's work (1914) on Rana pipiens, and especially by an extended series of contributions from Laurens (1914, 1915, 1916, 1917) on the larvae of several species of Amblystoma. Babak's views also received confirmation from the work of Fischel (1920) on various amphibians and especially from that of Duspiva (1931) on the two fishes already mentioned, Salmo salvelinus and Perca fluviatilis.

The references thus far cited pertain exclusively to oviparous fishes and amphibians. I know of only one writer who has recorded notes on the melanophore system of ovoviviparous vertebrates. In his paper on the genesis of chromatophores in fishes Eigenmann (1891) refers very briefly to the condition in the young of the ovoviviparous rockfish, Sebastodes, an embryo of which is figured by him. This small fish, when about to escape from the ovarian apparatus of the mother, is said by this author to have a very fully formed melanophore system whose functional possibilities, however, are not reported. Aside from this very meagre reference I know of no other on the melanophores in ovoviviparous vertebrates. It is therefore of interest to look into the condition of the newly-born pups of the smooth dogfish, Mustehis canis (Mitchill). This fish with its young can be had from time to time in the early summer at the Marine Biological Laboratory, Woods Hole, and I am under obligations to this Laboratory for the

COLOR CHANGES IN NEWLY-BORN DOGFISH

material on which the following observations were made. I am also indebted to the Woods Hole Oceanographic Institution for the use of their equipment in working on these fishes. My studies were made under a grant given me from the Milton Fund of Harvard University for the investigation of color changes in marine animals. To the administrators of this Fund I wish to express my sincere thanks for the aid generously extended to me.

OBSERVATIONS

The smooth dogfish of the New England coast releases its young, four to a dozen or more at a time, in early summer. The following observations were made either on young dogfishes born in the labora- tory tanks or on individuals removed from the females by what may be called a Csesarean operation. After the envelopes and umbilical vessels of these young fishes had been severed they could be removed and carried gently to the sea-water tanks. During this procedure they were as a rule perfectly passive. After they had been immersed in the sea water of the tank for a fraction of a minute or so, normal respiratory gill movements began and at about the same time swim- ming was initiated. Such newly-born fishes swam for a short time with somewhat unsteady equilibrium, but they soon gained in steadi- ness and mingled with others of their kind in the tank. At the time of these tests in June the young fishes were about 25 cm. in length, the largest measuring 33 cm.

A young dogfish when first taken from the uterus of the female, a dark situation, is slightly dark in tint. This indicates that the young fish is in what has been called in this paper the secondary phase of melanophore activity, that is, the phase in which the fish is dark in darkness and pale or dark in the light according to the environment. Whether the dogfish passes through an earlier primary phase while it is still in the uterus of the mother is unknown. More likely this stage has been omitted in this fish as it appears to have been in the amphib- ians Bombinator and Hyla (Babak, 1910) and in the fish Fundulus (Bancroft, 1912; Spaeth, 1913; WTyman, 1924; Gilson, 1926).

Newly-born dogfishes respond in tint very quickly to that of their environment. On June 4 seven dogfishes were born in one of the experimental tanks. Five hours after their birth four were put in a white-walled, illuminated tank and three in a similar black-walled one. Two hours later those in the black-walled tank were extremely dark and those in the white-walled one decidedly light (Fig. 2). Prepara- tions were made of the skin from an individual in each of these sets and microscopic views of the two preparations are shown in Figs. 3 and

4 G. H. PARKER

4. As might be expiated, the melanin in the color-cells from the pale dogfish is concentrated (Fig. 3), that from the dark one is dispersed (Fig. 4). On the following day the pale fishes were put into the black- walled tank and the dark ones into the white-walled one. Within two hours the fishes had reversed their tints, the pale ones having become dark and the dark ones pale.

In a second litter of dogfishes obtained by Caesarean operation three were put immediately after their removal from the uterus into a black-walled illuminated tank and three into a white-walled one. The three fishes destined for the black tank were put into it at 9:17 in the morning. By 9 :55 they had increased their dark tint. At 10 :30 they were dark but not fully so, and at 10:50 they were fully dark. The three dogfishes intended for the white tank were put into it at 9:20 in the morning. By 9:55 they had lightened considerably but were still somewhat dark. By 10:30 they were fully blanched as compared with other young dogfishes used as checks. From these two sets of tests it is fair to conclude that specimens of Mustelus immediately after birth are capable of responding to the tint of their environment by appropriate melanophore reactions. In these re- sponses they were quite like adults of their own species.

In another respect the young dogfishes also resembled adults. It has already been shown (Parker and Porter, 1934) that when a small transverse cut is made in the fin of a relatively dark Mustelus a. pale band is soon formed extending from the cut toward the free edge of the fin. This band is believed to result from an excessive stimulation of the concentrating melanophoric nerve-fibers cut by the operation. Similar conditions obtain in the newly-born dogfishes. The pectoral fins of two such fishes were cut as described and 25 minutes after the operation both fishes had well-developed pectoral bands. One of these fishes was killed and a preparation of its pectoral fin was made. A photograph of this fin is reproduced in Fig. 1 and shows the typical pectoral bands.

EXPLANATION OF PLATE

FIG. 1. Dorsal view of the left pectoral fin of a newly-horn Miistclus canis. Two small cuts transverse to the fin rays have been made, the anterior one nearer the root of the fin, the posterior one nearer its lateral edge. A pale band extends from near each cut to the light edge of the fin.

FIG. 2. Dorsal views of two newly-born Mustelus canis, the upper one in the dark condition, the lower one in the light condition. Preparations preserved in formaldehyde-alcohol.

FIG. 3. Microscopic view of the melanophores on the dorsal surface of the pectoral fin of a Mustelus canis in the light condition, melanin concentrated.

FIG. 4. A view similar to that shown in Fig. 3 of the fin of Mustelus canis in the dark condition, melanin dispersed.

6 G. H. PARKER

In a third respect newly-born dogfishes resemble adults. They respond in the same way to adrenalin and to pituitrin (Parke, Davis and Company's preparations) as the mature dogfishes do (Lundstrom and Bard, 19,32). If 0.2 cc. of a solution of adrenalin, one part in a thousand of water, is injected into a young dogfish of moderately dark tint, the animal will begin to blanch in about ten minutes and shortly after that it will assume a tint of extreme paleness. After two to three hours the young fish will reassume its darker tone. In a similar way an injection of 0.2 cc. of obstetrical pituitrin into a pale dogfish will induce its slow darkening. This change also passes away in about two hours after which the young dogfish will return to its former state. In both these respects the newly-born fishes resemble the adult.

These records show quite clearly that a young Mustelus immediately after birth possesses an active melanophore system whose reactions in a number of significant ways agree very fully with those of the adults. These ovoviviparous dogfishes then begin life in what has been called the secondary phase of melanophore activity. On the loss of their eyes they should lapse, according to the general theory of these rela- tions, to the condition of the primary phase. Unfortunately my material was not sufficiently abundant to allow this test to be made.

SUMMARY

1. Mustelus canis is an ovoviviparous dogfish in which the young are born with a body length up to 33 cm.

2. At birth the young dogfishes are of a moderately dark melano- phoric tint. This is doubtless the influence of the maternal body within which they have been lodged.

3. Immediately after birth these young dogfishes respond to their environment in that they change light or dark, conditions brought on by a concentration or a dispersion of their melanophore pigment.

4. Pale bands can be produced on the fins of newly-born Mustelus by cutting their nerves, as can be done with the adults.

5. A young Mustelus responds to injections of adrenalin by blanch- ing and to pituitrin by darkening as adults do.

6. A newly-born Mustelus shows no evidence of the primary phase of color change seen in some other fishes and in some amphibians. It appears to omit this phase in its ontogeny and is born with a melano- phore system that responds in the same way as this system does in the adults.

REFERENCES

BABAK, E., 1910. Zur chromatischen Hautfunkton der Amphihicn. Arch. ges.

Physiol., 131: 87. BANCROFT, F. \Y., 1912. Heredity of pigmentation in Fundulus hybrids. Jour.

Exper. Zoo/., 12: 153.

COLOR CHANGES IN NEWLY-BORN DOGFISH 7

MKCIIKR, II., 1929. Uber die Verwendung des Opak-IIluminators xu biologischen

Untersuchungen nehst Beobachtungen an den lebenden Chromatophoren

der Fischhaut im auffallenden I.ichl. Zeitschr. wiss. Mikr., 46: 89. DUSPIVA, F., 1931. Beitrage/.ur Physiologic der Melanophoren von Fischembryonen.

Sitzungsber. Akad. wiss. Wien, Math.-Naturwiss. Kl., 1, 140: 553. EIGENMA'NN, C. H,, 1891. On the genesis of the chromatophores in fishes. Am.

Nat., 25: 112. FISCHEL, A., 1920. Beitrage zur Biologie der Pigmentzelle. Anal. Hefte, Abt. Arb.,

58: 1. GILSON, A. S., JR., 1926. Melanophores in developing and adult Fundulus. Jour.

Exper. Zool., 45: 415. HOOKER, D., 1914. Amoeboid movement in the corial melanophores of Rana.

Am. Jour. Anal., 16: 237. LAURENS, H., 1914. The reactions of normal and eyeless amphibian larvae to light.

Jour. Exper. Zool., 16: 195. LAURENS, H., 1915. The reactions of the melanophores of Amblystoma larvae.

Jour. Exper. Zool., 18: 577. LAURENS, H., 1916. The reactions of the melanophores of Amblystoma larvae.

The supposed influence of the pineal organ. Jour. Exper. Zool., 20: 237. LAURENS, H., 1917. The reactions of the melanophores of Amblystoma tigrinum

larvae to light and darkness. Jour. Exper. Zool., 23: 195.

LUNDSTROM, H. M., AND P. BARD, 1932. Hypophysial control of cutaneous pig- mentation in an elasmobranch fish. Biol. Bull., 62: 1. PARKER, G. H., AND H. PORTER, 1934. The control of the dermal melanophores in

elasmobranch fishes. Biol. Bull., 66: 30. PERNITZSCH, F., 1913. Zur Analyse der Rassenmerkmale der Axolotl. I. Die

Pigmentierung junger Larven. Arch. mikr. Anat., 82: 148. SPAETH, R. A., 1913. The physiology of the chromatophores of fishes. Jour. Exper.

Zool., 15: 527. WYMAN, L. C., 1924. The reactions of the melanophores of embryonic and larval

Fundulus to certain chemical substances. Jour. Exper. Zool., 40: 161.

LIGHT INTENSITY AM) M I.I.AM >I'I K )RK RESPONSE

IN THE MINNOW, ERICYMBA

BUCCATA COPE

F. A. BROWN, JR. (From the Zoological Laboratory of the University of Illinois') l

Although every investigator of chromatic response in animals is well aware that light intensity has a profound effect upon the coloration of animals, this knowledge is at most qualitative and is in general restricted to the effects of the presence or absence of light. Fishes become pale in darkness as a rule while in light the response is governed by the color of the background. Considering that many animals with responsive pigmentary systems are normally accustomed to live at relatively low intensities of light, it was believed of significant value to investigate the behavior of fish from this viewpoint, and perhaps to derive simultaneously more information in regard to the sensitivity of the photoreceptors and the manner in which they function to produce adaptive color changes. In this latter regard Keeble and Gamble (1904), Bauer (1905), Sumner (1911), von Frisch (1911), Sumner and Keys (1929), Pearson (1930), and Sumner (1933) have concluded that the color of the animal is determined by the ratio of incident and reflected light striking the eye of the animal. Should this statement hold true without modification, then it would follow that for a given background which is characterized by always reflecting a given per- centage of the light striking it, the animal would remain the same shade at all effective light intensities and there would exist a sharp critical point in intensity of light at which a fish upon a black background would become pale as the light was decreased. Whether or not this is the actual state of affairs is tested in the following experiment.

I. UNIFORM BLACK BACKGROUND AND VARYING LIGHT

INTENSITY

Material and Methods

In the experiment herein described the silver-mouthed minnow, Ericymba buccata Cope, was used exclusively. This fish is from five to eight centimeters in length and is found most abundantly in streams with sand bottoms in the region about Urbana, Illinois, (Thompson and Hunt, 1930). The fish used were always freshly caught, none

1 Contribution No. 474.

8

LICHT INTENSITY AND MELANOP1 K >U K KKSPONSE

being used after they had been in the laboratory tanks for more than a week. Furthermore, all the data were obtained from fish caught in a given region and within a period of less than two winter months, thus insuring fish that were quite uniformly pigmented. Because of the rapidity of color change in this species no method was devised whereby more than a single light intensity could be used in testing the quantita- tive response in any individual fish. As a result, the data included here are those of a population and hence characterized by considerable variation.

All the experiments were carried out in an experimental dark-room. The light sources used in obtaining the different intensities were a 200 watt tungsten lamp, a 75 watt daylight Mazda lamp, and a 25 watt tungsten lamp with a daylight filter. For the lower intensities the last lamp was enclosed in a light-proof container possessing an iris diaphragm over the single aperture. In addition, approximately neutral light filters (exposed photographic plates) were used. The measurement of the light intensity was made with a potassium photo- electric cell with maximum sensitivity in the blue end of the visible spectrum. This cell was constructed by Professor J. Kunz. In nearly every instance the light source was 125 centimeters directly above the 8 inch, flat-black-coated crystallizing dish in which the experimental fish were placed.

In order to test the effect of a given light intensity upon the response of the melanophores, two or three minnows were placed in the crystal- lizing dish containing three centimeters of water. The crystallizing dish was coated with flat-black paint because it was determined that upon a background of this nature the fish would become dark in color in the ordinary light intensities of the laboratory and would become pale in total darkness. Thus the actual intensity at which the fish would entirely cease to respond to the color of the background could be learned. At the end of two to three hours in the experimental situation, during which time the water in which the fish were kept was constantly aerated with compressed air and an aerator block, the fish were removed and immersed in boiling water for a minute or two. Then a piece of the integument from a given area of the fish was dissected off and mounted in glycerine. A Spencer camera lucida and a celluloid rule served in measuring the diameters of 30 to 80 melanin masses. The average was taken as an index of the degree of dispersion of the pigment in the fish as a whole. In order to ascertain that two hours was sufficiently long for the pigment masses to have reached an equilibrium point of dispersion within the melanophores for the light intensity and background in question, some of the fish were taken from

10

F. A. BROWN, JR.

a black background in light and others from a white background in light at the beginning of the experiment and thus the equilibrium points were reached from both sides. Furthermore, at the intensities of the ordinary daylight of the laboratory the color-change was very rapid, requiring only five or ten minutes to pass from one extreme to the other. Research is now in progress to determine to what extent the rates of color change are influenced by intensity.

Results

During the course of the experimentation 75 minnows were tested. These were roughly distributed among the seven light intensities in

TABLE I

Average diameters of melanin masses in fish kept upon a black background under measured light intensities. Pigment mass diameters are given in micra and light intensity in foot candles.

Light intensity

.000005

.000053

.000625

.0125

.25

1.75

23.75

55.3

65.9

78.8

78.6

80.5

86.1

SS.5

52.5

48.4

68.3

76.9

78.5

84.4

82.0

40.5

43.7

63.4

74.0

76.3

83.4

80.3

40.4

41.6

58.3

72.5

64.3

81.4

80.2

36.6

38.2

56.4

68.6

54.0

80.5

79.8

33.9

39.3

55.0

67.6

74.5

77.1

68.6

36.3

51.9

59.6

73.6

77.0

66.7

34.3

51.1

63.5

72.5

76.6

34.0

44.0

54.3

69.1

75.3

40.7

41.9

53.5

59.7

74.7

41.4

38.9

51.3

57.5

64.3

34.0

49.3

54.6

46.9

46.3

45.6

36.6

Mean 43.1

H.I

53.4

59.2

67.9

78.3

78.0

proportion to the variability exhibited in the data. Table I is a sum- mary of the results.

The averages for a number of fish have been calculated for each light intensity. The values for individual fish are shown diagram- matically in Fig. 1, plotted against the logarithm of the light intensity. These data strongly suggest that the relationship is most probably one of a direct proportionality over the complete range of dispersion of pigment mechanically possible in the melanophore of this fish. In

LIGHT INTENSITY AND MELANOI'l K )RE RESPONSE

11

90

0

0

0

0

0 °

80

00 g

0

o o

o Q

o

8 8

.J

0 0

.E7°

o

0 ° 0 0

o

s

0

CO

o o

CO

0

o

E

60

0 0

c

o

o

£T

0 °

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o

o

8 3

o

o_

0

0

0 50

o

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8

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0

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0

'-13

0 °

QO 40

0 0

o^

0

2

0 0

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1

080

30

1 1 1

1 1 1 1

-5

-4-

-3 -2 -I

Logarithm of liqhf intensify

o

FIG. 1. Diagram showing the relationship between the logarithm of light intensity and the degree of melanin dispersion in the melanophores of Ericymba buccata. Each dot indicates the value for a single fish.

other words, the limits of this simple relationship are determined by the diameters of completely concentrated and fully dispersed pigment masses.

II. UNIFORM LIGHT SOURCE WITH DIFFERENT SHADES OF

GRAY BACKGROUNDS

Material and Methods

A second type of experiment was performed using the same material and methods as in the first, except that here the light was kept constant

12

F. A. BROWN, JR.

at 1.75 foot candles, the minimum intensity at which complete disper- sion of melanin could be obtained in response to a black background. Six backgrounds were used, the black one of the previous experiment, a white one, and four of intermediate shades of gray. The latter five were obtained by placing a glass crystallizing dish upon paper and cloth of the appropriate shades. The relative brightnesses of the direct light at the level of the background and the light reflected from the background were measured with a Macbeth illuminometer equipped with a brightness-determining device consisting of a trans-

TABLE II

Average diameters of melanin masses in fish kept upon black, white, and gray backgrounds in an incident light intensity of 1.75 foot candles. The ratio indicates

incident intensity

the - r^- 1 igment mass diameter is given in micra.

reflected intensity

Background ratio

2.54

6.60

12.4

35.4

140

201

37.2

38.2

35.6

5(,.()

75.7

86.1

31.6

39.2

35.0

45.3

73.6

84.4

35.7

36.2

40.0

42.7

70.7

83.4

37.9

40.9

68.0

81.4

40.3

40.7

63.0

80.5

32.3

41.4

57.1

77.1

46.4

55.7

77.0

74.5

77.8

76.6

59.5

74.8

75.3

37.3

74.7

38.2

64.3

46.7

48.6

Mean 35.8

37.9

36.9

47.5

68.6

78.3

lucent white plate. The values obtained serve best as comparative

direct light

ones. 1 he value ot the ratio, - ,. , , obtained tor the white

reflected light

background was 2.54; for the gray backgrounds, 6.60, 12.4, 35.4, and 140.; and for the black one, 201.

Results

The average diameters of the melanin masses in 45 fish kept upon these above-described backgrounds are recorded in Table II. The values for the black background have been taken from Part I of this paper. These results plotted as a dot diagram in Fig. 2 indicate that

LIGHT INTENSITY AND MELANOIM l< )K K RESPONSE 90

80

70

0

o

o o

o o

8

3 8

o

£60 o

o 6

E50

8 o

~5> E g

-040P0 °

o

o o

30 60 90 120 150 ISO 210

Incident intensitii Reflected intensity

~ _ incident light

r IG. z. Diagram showing the relationship between the ratio, - , and

reflected light

the degree of melanin dispersion in the minnow, Ericymba buccala. Each dot indi- cates the value for a single fish.

for a given light intensity the average diameter of the pigment within the melanophore is a direct function of the ratio of incident to re- flected light.

GENERAL CONSIDERATIONS

The ratio of incident to reflected light is thus seen to be inadequate to account completely for the degree of melanophore dispersion even

14 F. A. BROWN, JR.

when the intensity is considerably above the value of zero. Total intensity has a marked and significant effect. Duspiva (1931) found that light intensity had a very great effect upon the coloration of larvae of Perca fliiviatiUs, Sahno salvelinus, and some other fishes. Back- ground was without effect upon these forms. Roller (1934), however, reported that for larval Coregonus the background was the principal stimulus for melanophore activity while the light intensity had no influence until complete darkness was attained. These two papers are characteristic of the extremes in the literature regarding the effect of light intensity upon melanophores in fishes. The present research does not pretend to give a complete answer to this problem of long standing. Rather, it merely suggests that both forces are quite definitely influential in altering the coloration of fishes, but that the degree of overlapping of the two or the dominance of a single one determines the differences between individual species and even the same species at different developmental periods.

It is a well-known fact to fishermen of the Illinois River that when the water is laden with silt and consequently permits light to penetrate only a very short distance in any quantity, the fishes taken are pale in color, whereas in clear water the fishes are invariably dark in shade. If fishes swimming freely at some distance from the bottom in clear water can be figured as being upon the equivalent of a black back- ground since practically no light is reflected from below, then the silt in the more turbid waters can be conceived of as having effect through reduction of incident light and also augmentation of the reflected light entering the eyes of the fishes.

In closing, I wish to acknowledge my indebtedness to Dr. David H. Thompson of the Illinois Natural History Survey and to Professor J. Kunz of the Physics Department, University of Illinois, for generously supplying me with helpful suggestions and material during this investigation.

SUMMARY

1. A quantitative method of determining the influence of the environment upon the melanophores of small fishes is described.

2. The degree of dispersion of the melanin in (he melanophores of the silver-mouthed minnow, Ericymba buccata, is within certain limits determined by the total light intensity as well as by the shade of the background.

3. Upon a constant black background between the intensities of light, .000053 and 1.75 foot candles, the average diameter of pigment masses is directly proportional to the logarithm of the light intensity.

LIGHT INTENSITY AND MELANOPHORE RESPONSE 15

4. At an intensity of less than .000053 foot candles the fish are at their maximum degree of paleness in spite of a black background.

5. At 1.75 foot candles illumination the fish becomes maximally dark upon a black background, and the average diameter of melanin masses appears to vary in a directly proportional fashion with the

incident light , . , f . ,- ,

ratio. - . .. , -, which reaches the eyes ot the nsn.

reflected light

LITERATURE CITED

BAUER, V., 1905. Ueber einen objectiven Nachweis ties Simultankontrastes bei

Tieren. Zentralbl. f. Physiol., 19:453. DUSPIVA, F., 1931. Beitrage zur Physiologic der Melanophoren von Fischembry-

onen. Akad. Wiss. Wien, Math.-naturw. KL, Sitzungsber. Abt. 1, 140:

553. VON FRISCH, K., 1911. Beitrage zur Physiologic der Pigmentzellen in der Fischhaut.

Pfliiger's Arch.f. d. ges. Physiol., 138: 319. KEEBLE, F. W., AND F. W. GAMBLE, 1904. The colour-physiology of higher Crustacea.

Phil. Trans. Roy. Soc. London, B. 196: 295. ROLLER, G., 1934. Uber den Farbwechsel von Coregonenlarven. Biol. Zentralbl.,

54:419. PEARSON, J. F. W., 1930. Changes in pigmentation exhibited by the freshwater

catfish, Ameiurus melas, in response to differences in illumination. Ecology,

11: 703. SUMNER, F. B., 1911. The adjustment of flatfishes to various backgrounds: a study

of adaptive color change. Jour. Exper. Zool., 10: 409. SUMNER, F. B., AND A. B. KEYS, 1929. The effects of differences in the apparent

source of illumination upon the shade assumed by a flatfish on a given

background. Physiol. Zool., 2: 495. SUMNER, F. B., 1933. The differing effects of different parts of the visual field upon

the chromatophore responses of fishes. Biol. Bull., 65: 266. THOMPSON, D. H., AND F. D. HUNT, 1930. The fishes of Champaign County. Bull.

III. Nat. Hist. Surv., 19, Art. 1, 5.

RENAL FUNCTION IN MARINE TELEOSTS

II. THE NITROGENOUS CONSTITUENTS OF THE URINE OF

SCULPIN AND FLOUNDER, WITH PARTICULAR REFERENCE

TO TRIMETHYLAMINE OXIDE

ALLAN L. GRAFFLIN AND R. G. GOULD, JK.»

(From the Department of Anatomy, Harvard Medical School, and Department of Biochemistry, College of Physicians and Surgeons)

In view of the rather extensive work which has been done upon kidney function in the sculpin (Myoxocephalus octodecimspinosus), a common marine teleost (for review of literature see 34), it became of interest to investigate more fully the nitrogenous constituents of the urine. For comparison the flounder (Pseudopleuronectes americamis] was likewise studied. Analysis of samples of urine from these two species for the ordinary nitrogenous constituents showed that ap- proximately one-half of the total nitrogen was still unaccounted for. The presence of trimethylamine oxide in other marine fishes (see Table III) made it appear likely that this compound might be present in the urine of our animals. Investigation has demonstrated its occurrence in sculpin urine in significant amounts, but its absence in detectable quantities from the urine of the flounder. Brief supple- mental studies demonstrated the occurrence of the oxide in the urine of the daddy sculpin (Afyoxocephalus scorpius) and confirmed its presence in the urine of the goosefish (Lophius piscatorius] (18).

MATERIAL AND METHODS

Freshly caught sculpins and flounders were brought into the laboratory, the urinary papilla was tied off, and the fish were trans- ferred to live cars, usually for 24 hours, sometimes longer. At the end of this time they were killed by a blow on the head, and the urine was removed from the exposed bladder by syringe. Following are additional details concerning the urine samples listed in Tables I and II; unless otherwise noted preservation was by toluol and hydro- chloric acid. All samples were kept in a refrigerator when not in use. Sculpin 1: about 475 cc., from 217 sculpins; chloroform and toluol. Sculpin 2: about 475 cc., from 239 sculpins; 1 per cent sulfuric acid. Sculpin 3: 15 cc. from a few specimens. Flounder 1: about 475 cc., from 135 flounders; chloroform and toluol. Flounder 2: about 475 cc.,

1 With the assistance of Gordon Spence.

16

RENAL FUNCTION IN MARINE TELEOSTS

17

from 117 flounders; 1 per cent sulfuric acid. Flounder 3: 15 cc. from a few specimens. Flounder 4: same. Daddy sculpin: from two specimens. Goosefish: from one specimen in good condition. All sculpins and all but one group of fifty flounders (taken in net) were caught with hook and line. The urine samples were collected at the Mt. Desert Island Biological Laboratory, Salsbury Cove, Maine.

Total nitrogen was determined by the methods of Van Slyke (49) (Sculpin 1 and 2, Flounder 1 and 2) and Pregl (39) (all other specimens) ; ammonia by the method of Folin and Bell (13) ; urea by the method of Van Slyke (50), using 0.5 M. veronal rather than phosphate as a buffer because of the high magnesium content of the urine; uric acid by the method of Benedict and Franke (4) ; amino acids by the method of Folin (12); total creatinine by the method of Folin (11); and chloride by the method of Van Slyke (48) as modified by Smith (41). Tri- methylamine oxide was determined by the method of Hoppe-Seyler (24).

RESULTS

The analytical data for the ordinary nitrogenous constituents of the large pooled samples of sculpin and flounder urine are summarized in Table I. The chloride values for these samples, in millimols per

TABLE I

Distribution of nitrogenous constituents of sculpin and flounder urine.

Sculpir

L 1

Sculpin

2

Flounde

r 1

Flounde

r 2

Nitrogen