STUDIES IN COLOUR CHANGES OF FISH.

I .—THE ACTION OF CERTAIN ENDOCRINE SECRETIONS

IN THE MINNOW.*

BY H. R. HEWER, D.I.O, B.SC.

Plimmer Fellow in Pathology at the Imperial College of Science and Technology

WITH TWO PLATES.

I. Introduction2. Material and Methods3. Types of Chromatophores4. Normal Reactions5. Action of Adrenalin .6. Action of " Infundin " and

ary Extracts

•

Pituit-•

I.

CONTENTS.not133125126138130

131

7. Action of Pinealin and Thyroid

8. Controls

9. Conclusions and Summary

10. References . . . .

11. Description of Plates

Introduction.

FAGI

136'37137

139

'39

SINCE the discovery of the action of adrenalin upon the melano-phores of frogs, attention has been directed towards the effectsproduced by the secretions of the ductless glands upon thecolour changes of the lower vertebrates. In Amphibia thework of Hogben and Winton has done much to elucidate theproblem of colour change. This is all the more apparent, aspreviously all work had attempted to account for the variousphenomena by some nervous mechanism, and had produced somany conflicting evidences that the problem had seemedimpossible to unravel. In fish, however, the classical work ofPouchet, followed by the more recent researches of v. Frisch,has succeeded in demonstrating that the nervous system wasthe chief factor in the control of colour. This is, moreover,confirmed by the demonstration, by Ballowitz, of nerve endingson the melanophores. The many phenomena of colour changesin fish are so varied, however, that many are far from being

* Received September 26th, 1925.123

H. R. Hewerexplained by this work, and v. Frisch himself is fully awareof this.

It was for this reason, therefore, that attention has beenturned of late years to the possibility of some action of theinternally secreting glands. Spaeth has contributed much byhis work on isolated fish scales. His results showed thatadrenalin and the extract of the posterior lobe of the pituitaryboth cause contraction of the melanophores. This reactionto the pituitary extract is the opposite to that produced inAmphibia where expansion occurs.*

Recently, since the commencement of this research, Abolinhas published an account of his work in a similar directionon the local Viennese race of minnows. He finds that whileadrenalin causes contraction when injected in sufficient quantities,posterior lobe extract causes an expansion of the melanophoressuch as that produced by a black background. This apparentcontradiction of Spaeth's work would have been a sufficientstimulus for further investigation, had it not been that theauthor had already carried out experiments with results oppositeto those obtained by Abolin.

The present research was started to find out the effects ofthe internal secretions, injected into the living animal and toascertain the end-points of their actions, if any.

The objections to the method of injection into the wholeanimal are too well known to be repeated here. There are,however, in such a case as that under review, certain advantageswhich outweigh these objections. For instance, the reactionsin colour changes are generally acknowledged to be synchronous,and it would seem therefore to be more advantageous to treatwith the whole animal than with the isolated skin. An actionof a hormone on the blood-system, for example, would not havesufficient representation in isolated skin, and although it maybe proved eventually that such actions, if they exist, do nothave any effect during the normal existence of the animal, thesame may be said of peripheral actions which have beendiscovered by means of isolated portions. This does not, by

* A fall account of this and other work on colour change in fish may be foundin "The Pigmentary Effector System," by L. T. Hogben, Biologual Memoirs,Edinburgh.

" 4

Studies in Colour Changes of Fishany means, attempt to discredit the isolation method, which, asa matter of fact, is an essential to the complete elucidationof such a complex problem.

Abolin's work, therefore, does not definitely contradict thatof Spaeth". It presents a different aspect of it, and it wasthought worth while to reinvestigate the problem along thelines followed by Abolin in order to establish such a divergencebefore probing for the cause of it.

The author would like to take this opportunity to expresshis thanks to Prof. Mac Bride for much valuable advice andunder whose direction this work has been carried out in theZoological Laboratories of the Imperial College of Science andTechnology, South Kensington.

2. Material and Methods.

The material used for this research was all obtained througha dealer from one locality—a tributary of the Thames—and noracial differences have been observed.

Previous to their being used for experiments in the laboratory,the fish were kept in a fairly large tank with a gravel bottomplanted with weeds. A continuous flow of aerated water wasmaintained. Fish kept for any length of time before beingused for experiment were fed with house-fly larvae. It was notfound practicable to maintain a definite period between feedingand use, but such differences of time as occurred were not greatand did not seem to affect the results.

During the experiments the fish were kept in glass tankspainted half white and half black on the outside with a black-white partition to divide the two halves. This permitted thefish to be moved from one background to another withouthandling. There was a continuous flow of tap-water throughthe tanks, the overflow being led away by a system of syphons.

The amount of fluid injected was small owing to the sizeof the fish. Doses were given in o. i c.c. or 0.05 c.c. This ofcourse prevented such accuracy as could have been obtainedif larger doses had been used.

At the end of experiments the fish were killed and fixedwhole in 10 per cent, formol. This was done as quicklyas possible, so that a minimum of change in the state of

125

H. R. Hewerthe chromatophores occurred. For purposes of microscopicexamination pieces of skin were removed, cleared, and mounted.For the melanophores it was found necessary to remove skinfrom the whole side of a fish. This then included skin fromthe back, flank (barred portion), and the abdomen. They werecleared in xylol and mounted in balsam, all muscle beingremoved.

For examination of the erythrophores and xanthophores,glycerine was used as the clearing agent and glycerine jellyas the mounting medium. This was necessitated by the factthat the pigments contained by these cells are readily solublein alcohol and consequently these chromatophores becomeinvisible when treated in the usual way.

3. Types of Chromatophores.

In the minnow there are four types of chromatophores—melanophores, erythrophores, xanthophores, and iridophores.The melanophores are the most important factor in thenormal colour changes and will be dealt with in detail.

The melanophores are distributed over the head, back, andflank, being absent from the ventral surface of the abdomen andfrom the pectoral and pelvic fins. They are distributed in twolayers, both in the dermis. The upper layer is just below theepidermis while the lower layer lies below the scales (fig. 8).

In the back region the melanophores are small and lie fairlyclose together. They exhibit, moreover, a peculiar phase notfound in any other region of the body. In this phase themelanophores have a rounded appearance, but on closerexamination are found not to be contracted. The centre ofthe cell is less crowded with pigment granules than is theperiphery, and these granules are seen to be lying well upthe processes although closely packed together and havinga very definite line of demarcation from the rest of the process.The term " annulo-stellate" has been applied to this phase asit seems comparable to the " stellate" phase of the othermelanophores (fig. 7).

On the flank there appears to the naked eye a pattern ofdark bars and paler stripes. These bars are most markedin the dark phase of the fish, but they may be seen micro-

126

Studies in Colour Changes of Fishscopically in all phases. They appear to be due to differentialexpansion of the melanophores. This, however, does notentirely account for the phenomenon, as certain cases havebeen observed in which there was barring evident to thenaked eye, but all the melanophores were found to be in astate of complete expansion. There must be, therefore, somenumerical differentiation as well. The differential expansionis most marked in the lower layer, although it is also presentin the upper layer but to a less extent. The barring extendsboth above and below the lateral line so that the melanophoresof the back region, distinguished by their smaller size, are alsoaffected in the above manner.

In some cases there are melanophores present at the sideof the abdomen. These, when present, are very large and liein a single layer corresponding to the lower layer elsewhere.

Within the foregoing types there may be found considerablevariations in size. In general there are two kinds, large andsmall. That this is any true or fundamental difference mayvery well be doubted, for intermediate forms were found andno physiological differences have been noticed.

The question of the method by which the melanophores"expand ' and "contract" cannot be entered into here. Theauthor thinks, however, that the weight of the evidence goes toshow that a movement of the granules only takes place. Theterms "expand" and "contract" have, however, become sousual in this connection that they will be used here withoutreference to the actual mode of reaction.

For the accurate estimation of the phase of the melanophoresvarious stages were recognised, and the following terms wereapplied: (i) Contracted; (2) slightly stellate; (3) stellate;(4) slightly expanded; (5) expanded; (6) fully expanded.Type conditions are given in figs 1-6. Besides these, oneother was established and known as "annulo-stellate." Adescription of this has been given above.

The xanthophores are distributed over the same areas asthe melanophores. No differentiation has been observed otherthan a distribution in two layers.

Erythrophores are not found in all minnows. When theydo occur they are usually present in the following areas:

127

H. R. Hewer(i) At the base of the fins; (2) over the eyes; (3) above andbehind the operculum; (4) on the lips; and (5) in theabdominal-branchial region, sometimes joining up with thepatches at the base of the pectoral and pelvic fins, butusually and most markedly along the mid-ventral line. Theirpresence is most marked during the breeding season, andappears to be due to an increase in numbers rather than to anypermanent phase of expansion.

The xanthophores and erythrophores seem to be closelyrelated as their reactions are identical, and the pigments ofboth are soluble in alcohol and acetone and other solvents.Intermediate forms which are orange in colour have been foundin the flank region where the xanthophores give place to theerythrophores of the abdominal region. This may indicatea closer relationship than might be supposed from any chemicalsimilarities.

For purposes of tabulation of the phases of the xanthophoresand erythrophores, three type stages were established, namely:(1) Contracted; (2) stellate; (3) expanded. They correspondroughly with the same stages of the melanophores.

The iridophores were apparently always in the expandedcondition and were situated in the flank and abdominalregions, and on the operculum. The whiteness and iridescenceof certain parts are due to their presence in small or greatnumbers. They do not form any conspicuous pattern andseem to act merely as a background or reflecting layer for theincident light.

4. Normal Reactions.The normal reactions of the minnow were not fully investi-

> gated as a considerable amount of work has been done onthis already, and only those observations were made whichwere necessary for comparison with the experimental results.Lighting and temperature were fairly constant, so that theeffects of background only were observed.

Melanophores.—Little or no difference could be seen betweenthe two layers of melanophores when the fish were placedon a white background. The average phase was that of"slightly stellate." "Contracted" and "stellate" phases were

128

Studies in Colour Changes of Fishpresent in some cases although the latter was more infrequent.In the flank region traces of bars were observed microscopically.On the bars the melanophores were slightly more stellate thanelsewhere.

On a black background the most prominent feature was thebarred effect, and it became necessary to classify the resultsaccording to the pattern. The average results were asfollows: The melanophores of the bars were always "fullyexpanded" in both layers. On the stripes (the paler areasbetween the bars), the melanophores were always less expandedthan on the bars. The average phase was " expanded" inthe upper layer and " slightly expanded " in the lower layer. Insome cases the " stellate " phase was present in the lower layer.In the back region the upper layer of melanophores was "fullyexpanded," but the lower layer showed an average value of"expanded" varying from "fully expanded" to "slightlyexpanded."

In the dark there is again the appearance of barring butnot to the same extent as on a black background. The results,moreover, are not so concordant. In the upper layer thereis a general tendency towards expansion. The average valuesare : bars, " expanded " ; stripes, " expanded " ; back, " stellate "to "fully expanded." The last value is essentially vague, ashalf are "stellate" and half are "fully expanded." In the lowerlayer there is a falling off of the tendency to expand as shownby the following average phases: bars, "expanded"; stripes,"stellate"; back, "stellate." The peculiar phase occurring inthe back region • is here very evident and is marked as theaverage phase.

The normal reactions of the melanophores in general (atmedium temperature) are therefore: Contraction on a whitebackground and expansion on a black background ; in the darkthere is a general tendency to expand.

These reactions are very rapid if compared with those ofthe frog. Considerable individual variations exist but usuallythe time of reaction is about five or six minutes. This observa-tion is, of course, made by the naked eye, and it is probablethat the reaction is completed a short time after this. In thesame way the reaction does not appear to start for one or

129

H. R. Hewertwo minutes. In the above experiments the fish were alwaysleft under the various conditions for at least three hours, andin many cases for longer. Those in the dark were left forfive hours.

Xanthophores and Eiythrophores-—The reactions of thesetwo types of chromatophores may be taken together as theyare identical.

On a white background these chromatophores always are inthe "contracted" phase. On a black background, however,the results vary from "contracted" to "expanded." Thisrange of phases may be seen on one and the same specimenand is not due to individual variability of reaction. The onlymethod that seems feasible for stating their reactions is to saythat they are neutral to black background, but that a whitebackground acts as a strong stimulus for contraction.

5. Action of Adrenalin.

The action of adrenalin has always been stated to be that ofcausing contraction of the melanophores, whether injected intothe living animal or applied to the isolated scales. In thepresent research this has been confirmed. Solutions weremade up in a Ringer solution approximately isotonic withthe blood of fresh-water fish. Injections were made onminnows on a black background, they were therefore in thedark phase. All injections were made intramuscularly, intra-peritoneal ones having no effect whatever. All the fish werekilled fifteen minutes after the injections.

Doses of 0.05 mgm., 0.01 mgm., and 0.005 mgm. causea contraction of the melanophores to take place over all thebody. This pallor is very intense and frequently exceedsthe average state on a white background. A dose of 0.001mgm. causes paling which is more or less localised on the sideof injection. The paling makes itself apparent first around thepoint of injection, and afterwards spreads over the side. 0.0005mgm. only causes limited local pallor, while doses of 0.0001mgm. and 0.00005 mgm. produce very local and transitorypaling. Smaller doses evoke no change whatever in colour-Injections of 0.1 c.c. of the Ringer similarly produce no result.

The local action is very sharply marked as may be seen in130

Studies in Colour Changes of Fishfig. 16. Where it is present, it is very intense and differs tono appreciable extent from the general pallor produced by thelarger doses. There is no reason to suppose that the generaland local effects are different in origin and may therefore be thesame as that found in isolated scales treated with adrenalin.The minimal dose is therefore 0.00005 mgm. and the sub-minimal 0.00001 mgm.

The following is a summary of results :—

Dose.

0-05 mgm.001 „

0005 „

O-OOI „

0 0 0 0 5 .»

OOOOI „0-00005 »O-OOOOI „O-OOOOO5 „

o-i cc. Ringer

Macroscopic Results.

Rapid intense pallorIntense pallor

n

Paling localised on side ofinjection

Paling locally round pointof injection

Slight paling locally

No changent t

Microscopic Results.

Contracted

Slightly stellate

Local. General.Contracted

»

Stellate

Normal

»

!»

Normal»i t

The effect on the erythrophores is similar; contractionbeing produced. The xanthophores react in exactly the sameway as the erythrophores. The end-points of both xantho-phores and erythrophores are difficult to state accurately astheir reaction to black ground is neutral. It is therefore possibleto obtain contracted chromatophores in sub-minimal doses.With these limits then it may be stated that the end-pointswere the same as for the melanophores.

6. Action of "Infundin" and Pituitary Extracts." Infundin."—This sterile extract was used for the first series

of experiments. The solutions were made up in the sameRinger as that employed for the adrenalin experiments.

On a White Background. — The following doses wereadministered: 0.01 c c , 0.005 c-c-» 0.001 c c , 0.0005 c c ,0.0001 c c , 0.00005 c-c-> 0.00001 c c , and 0.000005 c-c- °f t n e

sterile extract (a 17 per cent, solution) ; and o. 1 c c Ringer.While on macroscopic examination it was thought that in

the higher doses a slight flush was produced in the dorsal

H. R. Hewerregion, it was found to be quite transitory and was probably theeffect of moving and handling during injection. Microscopicallythere was not found to be any reason to suppose that " Infundin "produced any darkening (expansion) comparable to that statedby Abolin. In some cases the melanophores showed a tendencytowards expansion, but this did not exceed what might beexpected from individual differences. Moreover, there was notany agreement among these cases as to dosage.* The Ringerproduced no effect on the colour.

The erythrophores and xanthophores were both expandedto a "stellate" or an "expanded" condition by these injections.The minimal dose is o.ooi c.c. (The normal phase iscontracted.)

On Black Background.—The same range of doses wasemployed. The only effect which was observed macroscopicallywas a paling in the dorsal region in the two largest doses.Microscopically this was shown to be confined to the lowerlayer of melanophores which were in the " stellate " conditionalthough several cases showed even further contraction. Thoseinjected with o.ooi c.c. showed slightly less contraction thanthis, and the lower doses produced no deviation from the normalcondition on black background. The Ringer produced no effect.

The erythrophores and xanthophores were consistentlystellate where doses of o.oi c.c., 0.005 ac-> o r 0.001 c.c. wereemployed. Below 0.001 c.c. they varied between contractedand expanded—the normal reaction to a black background.

As Abolin had employed distilled water to make upthe " Infundin" solutions the previous experiments are notrepetitions of his work. Injections of 0.01 c.c. sterile extractmade up in distilled water were, therefore, injected into paleminnows on a white background. The injections were madeintramuscularly and intraperitoneally, but in neither case couldany expansion of the melanophores be observed. The erythro-phores and xanthophores gave the same reactions as wereobserved above when Ringer solution was employed. Injectionsof o. 1 c.c. of distilled water produced a slight irregular darkeningwhen administered intramuscularly, but no effect was producedwhen injected intraperitoneally.

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H. R. HewerNo confirmation of Abolin's work was obtained therefore in

these experiments. In fact, a reaction which resembled thatobtained by Spaeth was observed. To test this further thefollowing experiments were carried out.

Injections of Extract of Cod's Pituitary.—Extracts of freshcod's pituitary were made up in Ringer and injected, into darkminnows kept on a black background. The doses are given ingrammes weight of the fresh gland (whole).

°-°°3 g"1-* o.ooi gm., and 0.0005 Sm- caused completepallor over all the body; and on microscopic examination themelanophores were found to be in the phases of "contracted"or " slightly stellate." The upper layer of melanophores, in thecase of the dose of 0.0005 Sm-> were> however, slightly lesscontracted, being in the phase of "stellate" for the most part.

0.0001 gm., 0.00005 gm>> a n d 0.00001 gm. all showed tracesof local pallor, but this did not last long and disappeared beforethe fish were fixed so that microscopic examination only showsthe normal reaction to black background.* The fish used inthe 0.003 Sm' dosage were killed and fixed ten minutes afterinjection. The rest were fixed half an hour after injection.

That the active hormone was present in the posterior lobe,and was therefore probably identical with the hormoneproducing pallor in " Infundin," is illustrated by the followingexperiment.

One cod's pituitary was roughly f separated into its twolobes. Extracts were made up of approximately equal strengthsof the two parts by weight. The following injections weremade:—

Posterior Lobe Extract.

0.5 c.a into a pale frog . . Darkening evident after 15 minutes.0.1 c c into two minnows . General pallor evident after a few minutes.

Anterior Lobe Extract.

0.5 c c . into a pale frog . . Slight darkening after 20 minutes,a 1 c c into two minnows . Local pallor after a few minutes, which

spread somewhat in one fish.

• See Table II. at p. 135.+ The pituitary of the cod is rather complicated in its structure, and consequently

considerable difficulty was experienced in completely separating the two lobes.Diffusion of hormone must certainly have taken place previous to separation so thatallowance for this source of error most be made as welL

134

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H. R. Hewer" I nfundin" extracts were subjected to tests by peptic and

tryptic digestion, and it was found that the hormone producingthe foregoing results behaves in the same way as the one whichacts on the melanophores of the frog. This points to apossible identity of the two hormones. If that is the case, thenit constitutes another point in favour of the view that themelanophores of fish and amphibia are fundamentally differentphysiologically.

It may be stated then that in the minnow a hormone,present in the extract of posterior lobe of the pituitary, causespallor and is possibly the same as that causing expansion of themelanophores in the amphibia. The minimal doses for pro-ducing this reaction are o.ooi c.c. in the case of " Infundin "and 0.0005 gm. of the fresh cod's pituitary.

On the erythrophores and xanthophores uniform expansionis produced by posterior lobe extracts. The minimal dose for"Infundin" is about 0.001 c.c, while for an extract of cod'spituitary 0.0005 gm. of the fresh gland is the minimum.

7. Action of Pinealin and Thyroid.

The following injections were made with pinealin but, aswill be seen, no change in colour was evoked :—

Intramuscular Injection.

(a) 1 c.c. of a A gr. in 10 c.c. Ringer solution was injectedinto two dark minnows on a black background. Nochange.

(5) 1 c.c. of a -rV gr. in 10 c.c. Ringer solution was injectedinto two pale minnows on white background. Nochange.

Intraperitoneal Injection.

(c) 0.1 c.c. of a ^ gr. in 10 cc. Ringer solution was injectedinto two dark minnows on a black background. Nochange.

(d) o.ic.c. ofa A gr. in 10c.c. Ringer solution was injectedinto two pale minnows on a white background. Nochange.

136

Studies in Colour Changes of FishSimilar injections were made with thyroid extract but they

also were without effect. In all cases the same Ringer wasused to make up the solutions.

a Controls.Certain other tissues were also extracted and injections

made. Extracts of ox liver and muscle were made up andinjected into pale and dark minnows. In the pale fish therewas no change, but in the dark ones there occurred slight localpallor which was transitory.

Extracts of muscle and brain from a haddock were made upto a 5 per cent, solution and o.i c.c. injected into dark minnows.Apart from slight transitory local paling there was no changefrom the normal.

9- Conclusions and Summary.

The results obtained in this paper constitute a directnegation of the work of Abolin in the case of " Infundin"injection unless racial differences exist. It is extremely unlikely,however, that any such differences would account for a dia-metrically opposite result. Moreover, the results here enumeratedagree with those of Spaeth. It was pointed out in the intro-duction that there may be differences between reactions inducedby injections into the whole animal and those produced onisolated scales or portions of skin. The fact of identicalreactions, however, suggests that in this case the method ofreaction may be the same. This point has not been dealt with,as the reasons set forth below indicate that post-pituitaryextract does not play any important part in the normal colourchanges of the minnow.

The action of adrenalin appears to be peripheral whetherinjected or applied to isolated scales. The local and generalreactions are merely due to differences in dosage. This beingthe case, the minimal dose, while being the smallest dose toproduce contraction of the melanophores, is actually merelya gauge of concentration. The figure which is of importanceis really that which gives the " minimal concentration " over thewhole animal, producing a pallor comparable to that found inthe normal reaction to a white background.

VOL. III.—NO. 2. 137 13

H. R. HewerAs regards the effects which the results of this research may

have on the general theory of colour changes in fish, it mustbe pointed out that of the four hormones investigated, only two,namely adrenalin and post-pituitary extract, have any effect oncolour. Furthermore, both these hormones produce a con-traction of the melanophores. Thus there are none of thesefour which produce expansion.

Whether adrenalin or post-pituitary extract produce thecontraction in the normal conditions is the next problem. Itdoes not seem probable, to the author, that post-pituitaryextract could act in this way in the normal anomal. Thiswould appear for two reasons. Firstly, the amount for pro-ducing contraction is extraordinarily large. The amount of"Infundin" required to produce contraction in a minnow isabout ten times greater than that required to produce expansionin a frog, an animal a little more than ten times as large.Furthermore, the minimal dose of cod's pituitary for a minnowis 0.0005 Sm-> a n d the whole gland varies in weight from o. 1 gm.to 0.05 gm. depending on the size of the cod. This means that,taking the gland as weighing 0.1 gm., there is only enoughfor paling two hundred minnows, assuming that the sensitivityof the melanophores and the concentration of the hormone inthe gland are the same in both cod and minnow. Comparisonof weights of the animals shows that the amount of hormonepresent would not have any effect in normal reactions.

Secondly, it will be observed that above the minimal doserequired to produce contraction of the melanophores, expansionof the erythrophores occurs. Now the normal condition is thatif the melanophores are contracted, then the erythrophores arealso contracted. If, however, post-pituitary extract is themeans of producing contraction of the melanophores, thenexpansion of the erythrophores would be expected to accompanyit. So it will be seen that the artificial conditions do notcoincide with the normal.

Any action, on the part of post-pituitary extract, on theerythrophores may also be doubted- For the action of thisextract is to produce uniform expansion, and it has been shownthat under the normal conditions of a black background ery-throphores in all phases may be seen in one and the same

138

Studies in Colour Changes of Fishindividual. Further, such expansion is accompanied by con-traction of the melanophores, which does not occur on a blackbackground. It must, therefore, be admitted that post-pituitaryextract does not take part in the normal reactions of eitherthe melanophores or erythrophores. The same applies to thexanthophores, which are identical in their reactions with theerythrophores.

It is not proposed here to deal with adrenalin further thanhas already been done. The reason for this may be brieflygiven. Adrenalin acts either on the nerve endings or onthe melanophores themselves, since its action is peripheral.The former is the more likely as it is its usual seat of action.This brings the problem within the range of the nervous controlof colour change, and it becomes impossible to deal with anyone type of reaction without an extensive consideration of manyother actions. This will, therefore, be dealt with at somefurther date.

10. References.Abolin, L. (1925), Archiv.f. Mikro. Anat. u. Entw.-mech., 104, 667-698.Ballowiti, E. (1893), Ztschr.f. wiss. Zool., 68, 673-706.v. Frisch, K. (1911), PftUgtrs Arch^ 188, 319-387.v. Frisch, K. (1911), Biol. CentraliL, 81, 236-248 and 412-415.v. Frisch, K.-(i9i2), Zool. Jakr., 82, 171.23aPouchet, M. G. (1872), Journ. de PAnat. et de la Pkysiol^ etc., 8, 71-74.Pouchet, M. G. (1871), Compttt Rtndues (Bu>logie\ p. 113.Spaeth, R. A. (1913), Journ. Exp. Zool., 10, 537-579.Spaeth, R. A. (1916), Journ. Exp. Zool^ 20, 193-215.Spaeth, R. A., and Barboor, H. G. (1917), Journ. Pharm. and Exp. Ther^ 9,

43' -44°.For further references see footnote p. 124. Also Fuchs in Handbuch d. Verglei-

chtnden Phytiologie, Hans Winterstein, Jena, which gives a complete list of literatureup to 1914.

Description of Plates.FIGS. 1-7.—Melanophores in various typical phases.

1. "Contracted."2. " Slightly stellate."3. "Stellate."4. " Slightly expanded."5. "Expanded."6. " Fully expanded."7. " Annulo-stellate." This melanophore is from the back region and

is magnified twice as much as the others.Fio. 8.—Longitudinal section of the skin, showing position of dermal melanophores

in relation to epidermis and scales. Melanophores as large black masses.139

H. R. HewerFIG. 9.—Photomicrograph showing condition of the melanophores of a minnow

on a white background.FIG. 10.—Photomicrograph showing condition of the melanophores of a minnow

on a black background.FIG. 11.—Photomicrograph to show the barred effect produced by differential

expansion of the melanophores in the flank region. Note position of lateralline as indicated by the blood-vessel.

FIG. 12.—Photomicrograph to show the effect of "Infundin" injected into paleminnows on a white background. Compare with fig. 9.

FIG. 13.—Photomicrograph to show effect of an injection of an extract of cod'spituitary into a dark minnow on a black background. Compare with fig. 10.

FIG. 14.—Photomicrograph to show effect of "Infundin" injected into a darkminnow on a black background. Compare with fig. 10.

FIG. 15.—Photomicrograph to show the effect of adrenalin. This is the generalaction. Injected into a dark minnow on a black background.

FIG. 16.—Photomicrograph to shbw the effect of adrenalin. The effect here islocalised. Injected into a dark minnow on a black background.

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