I . — T H E 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. Introduction
2. Material and Methods 3. Types of Chromatophores 4. Normal Reactions
5. Action of Adrenalin . 6. Action of " Infundin " and
ary Extracts
•
Pituit-•
I.
CONTENTS. not
133 125 126 138 130
131
7. Action of Pinealin and Thyroid 8. Controls
9. Conclusions and Summary 10. References . . . . 11. Description of Plates
Introduction.
FAGI
136 '37
137 139
'39
SINCE the discovery of the action of adrenalin upon the
melano-phores of frogs, attention has been directed towards the effects produced by the secretions of the ductless glands upon the colour changes of the lower vertebrates. In Amphibia the work of Hogben and Winton has done much to elucidate the problem of colour change. This is all the more apparent, as previously all work had attempted to account for the various phenomena by some nervous mechanism, and had produced so many conflicting evidences that the problem had seemed impossible to unravel. In fish, however, the classical work of Pouchet, followed by the more recent researches of v. Frisch, has succeeded in demonstrating that the nervous system was the chief factor in the control of colour. This is, moreover, confirmed by the demonstration, by Ballowitz, of nerve endings on the melanophores. The many phenomena of colour changes in fish are so varied, however, that many are far from being
* Received September 26th, 1925.
explained by this work, and v. Frisch himself is fully aware of this.
It was for this reason, therefore, that attention has been turned of late years to the possibility of some action of the internally secreting glands. Spaeth has contributed much by his work on isolated fish scales. His results showed that adrenalin and the extract of the posterior lobe of the pituitary both cause contraction of the melanophores. This reaction to the pituitary extract is the opposite to that produced in Amphibia where expansion occurs.*
Recently, since the commencement of this research, Abolin has published an account of his work in a similar direction on the local Viennese race of minnows. He finds that while adrenalin causes contraction when injected in sufficient quantities, posterior lobe extract causes an expansion of the melanophores such as that produced by a black background. This apparent contradiction of Spaeth's work would have been a sufficient stimulus for further investigation, had it not been that the author had already carried out experiments with results opposite to those obtained by Abolin.
The present research was started to find out the effects of the internal secretions, injected into the living animal and to ascertain the end-points of their actions, if any.
The objections to the method of injection into the whole animal are too well known to be repeated here. There are, however, in such a case as that under review, certain advantages which outweigh these objections. For instance, the reactions in colour changes are generally acknowledged to be synchronous, and it would seem therefore to be more advantageous to treat with the whole animal than with the isolated skin. An action of a hormone on the blood-system, for example, would not have sufficient representation in isolated skin, and although it may be proved eventually that such actions, if they exist, do not have any effect during the normal existence of the animal, the same may be said of peripheral actions which have been discovered by means of isolated portions. This does not, by
* A fall account of this and other work on colour change in fish may be found in "The Pigmentary Effector System," by L. T. Hogben, Biologual Memoirs, Edinburgh.
Studies in Colour Changes of Fish
any means, attempt to discredit the isolation method, which, as a matter of fact, is an essential to the complete elucidation of such a complex problem.
Abolin's work, therefore, does not definitely contradict that of Spaeth". It presents a different aspect of it, and it was thought worth while to reinvestigate the problem along the lines followed by Abolin in order to establish such a divergence before probing for the cause of it.
The author would like to take this opportunity to express his thanks to Prof. Mac Bride for much valuable advice and under whose direction this work has been carried out in the Zoological Laboratories of the Imperial College of Science and Technology, South Kensington.
2. Material and Methods.
The material used for this research was all obtained through a dealer from one locality—a tributary of the Thames—and no racial 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 bottom planted with weeds. A continuous flow of aerated water was maintained. Fish kept for any length of time before being used for experiment were fed with house-fly larvae. It was not found practicable to maintain a definite period between feeding and use, but such differences of time as occurred were not great and did not seem to affect the results.
During the experiments the fish were kept in glass tanks painted half white and half black on the outside with a black-white partition to divide the two halves. This permitted the fish to be moved from one background to another without handling. There was a continuous flow of tap-water through the tanks, the overflow being led away by a system of syphons.
The amount of fluid injected was small owing to the size of the fish. Doses were given in o. i c.c. or 0.05 c.c. This of course prevented such accuracy as could have been obtained if larger doses had been used.
At the end of experiments the fish were killed and fixed whole in 10 per cent, formol. This was done as quickly as possible, so that a minimum of change in the state of
the chromatophores occurred. For purposes of microscopic examination pieces of skin were removed, cleared, and mounted. For the melanophores it was found necessary to remove skin from the whole side of a fish. This then included skin from the back, flank (barred portion), and the abdomen. They were cleared in xylol and mounted in balsam, all muscle being removed.
For examination of the erythrophores and xanthophores, glycerine was used as the clearing agent and glycerine jelly as the mounting medium. This was necessitated by the fact that the pigments contained by these cells are readily soluble in alcohol and consequently these chromatophores become invisible 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 the normal colour changes and will be dealt with in detail.
The melanophores are distributed over the head, back, and flank, being absent from the ventral surface of the abdomen and from the pectoral and pelvic fins. They are distributed in two layers, both in the dermis. The upper layer is just below the epidermis while the lower layer lies below the scales (fig. 8).
In the back region the melanophores are small and lie fairly close together. They exhibit, moreover, a peculiar phase not found in any other region of the body. In this phase the melanophores have a rounded appearance, but on closer examination are found not to be contracted. The centre of the cell is less crowded with pigment granules than is the periphery, and these granules are seen to be lying well up the processes although closely packed together and having a very definite line of demarcation from the rest of the process. The term " annulo-stellate" has been applied to this phase as it seems comparable to the " stellate" phase of the other melanophores (fig. 7).
On the flank there appears to the naked eye a pattern of dark bars and paler stripes. These bars are most marked in the dark phase of the fish, but they may be seen
scopically in all phases. They appear to be due to differential expansion of the melanophores. This, however, does not entirely account for the phenomenon, as certain cases have been observed in which there was barring evident to the naked eye, but all the melanophores were found to be in a state of complete expansion. There must be, therefore, some numerical differentiation as well. The differential expansion is most marked in the lower layer, although it is also present in the upper layer but to a less extent. The barring extends both above and below the lateral line so that the melanophores of the back region, distinguished by their smaller size, are also affected in the above manner.
In some cases there are melanophores present at the side of the abdomen. These, when present, are very large and lie in a single layer corresponding to the lower layer elsewhere.
Within the foregoing types there may be found considerable variations in size. In general there are two kinds, large and small. That this is any true or fundamental difference may very well be doubted, for intermediate forms were found and no physiological differences have been noticed.
The question of the method by which the melanophores "expand ' and "contract" cannot be entered into here. The author thinks, however, that the weight of the evidence goes to show that a movement of the granules only takes place. The terms "expand" and "contract" have, however, become so usual in this connection that they will be used here without reference to the actual mode of reaction.
For the accurate estimation of the phase of the melanophores various stages were recognised, and the following terms were applied: (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, one other was established and known as "annulo-stellate." A description of this has been given above.
The xanthophores are distributed over the same areas as the melanophores. No differentiation has been observed other than a distribution in two layers.
Erythrophores are not found in all minnows. When they do occur they are usually present in the following areas:
(i) At the base of the fins; (2) over the eyes; (3) above and behind the operculum; (4) on the lips; and (5) in the abdominal-branchial region, sometimes joining up with the patches at the base of the pectoral and pelvic fins, but usually and most markedly along the mid-ventral line. Their presence is most marked during the breeding season, and appears to be due to an increase in numbers rather than to any permanent phase of expansion.
The xanthophores and erythrophores seem to be closely related as their reactions are identical, and the pigments of both are soluble in alcohol and acetone and other solvents. Intermediate forms which are orange in colour have been found in the flank region where the xanthophores give place to the erythrophores of the abdominal region. This may indicate a closer relationship than might be supposed from any chemical similarities.
For purposes of tabulation of the phases of the xanthophores and erythrophores, three type stages were established, namely: (1) Contracted; (2) stellate; (3) expanded. They correspond roughly with the same stages of the melanophores.
The iridophores were apparently always in the expanded condition and were situated in the flank and abdominal regions, and on the operculum. The whiteness and iridescence of certain parts are due to their presence in small or great numbers. They do not form any conspicuous pattern and seem to act merely as a background or reflecting layer for the incident light.
4. Normal Reactions.
The normal reactions of the minnow were not fully investi-> gated as a considerable amount of work has been done on
this already, and only those observations were made which were necessary for comparison with the experimental results. Lighting and temperature were fairly constant, so that the effects of background only were observed.
Melanophores.—Little or no difference could be seen between
the two layers of melanophores when the fish were placed on a white background. The average phase was that of "slightly stellate." "Contracted" and "stellate" phases were
Studies in Colour Changes of Fish
present 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 than elsewhere.
On a black background the most prominent feature was the barred effect, and it became necessary to classify the results according to the pattern. The average results were as follows: The melanophores of the bars were always "fully expanded" in both layers. On the stripes (the paler areas between the bars), the melanophores were always less expanded than on the bars. The average phase was " expanded" in the upper layer and " slightly expanded " in the lower layer. In some cases the " stellate " phase was present in the lower layer. In the back region the upper layer of melanophores was "fully expanded," but the lower layer showed an average value of "expanded" varying from "fully expanded" to "slightly expanded."
In the dark there is again the appearance of barring but not to the same extent as on a black background. The results, moreover, are not so concordant. In the upper layer there is a general tendency towards expansion. The average values are : bars, " expanded " ; stripes, " expanded " ; back, " stellate " to "fully expanded." The last value is essentially vague, as half are "stellate" and half are "fully expanded." In the lower layer there is a falling off of the tendency to expand as shown by the following average phases: bars, "expanded"; stripes, "stellate"; back, "stellate." The peculiar phase occurring in the back region • is here very evident and is marked as the average phase.
The normal reactions of the melanophores in general (at medium temperature) are therefore: Contraction on a white background and expansion on a black background ; in the dark there is a general tendency to expand.
These reactions are very rapid if compared with those of the frog. Considerable individual variations exist but usually the time of reaction is about five or six minutes. This observa-tion is, of course, made by the naked eye, and it is probable that the reaction is completed a short time after this. In the same way the reaction does not appear to start for one or
two minutes. In the above experiments the fish were always left under the various conditions for at least three hours, and in many cases for longer. Those in the dark were left for five hours.
Xanthophores and Eiythrophores-—The reactions of these
two types of chromatophores may be taken together as they are identical.
On a white background these chromatophores always are in the "contracted" phase. On a black background, however, the results vary from "contracted" to "expanded." This range of phases may be seen on one and the same specimen and is not due to individual variability of reaction. The only method that seems feasible for stating their reactions is to say that they are neutral to black background, but that a white background acts as a strong stimulus for contraction.
5. Action of Adrenalin.
The action of adrenalin has always been stated to be that of causing contraction of the melanophores, whether injected into the living animal or applied to the isolated scales. In the present research this has been confirmed. Solutions were made up in a Ringer solution approximately isotonic with the blood of fresh-water fish. Injections were made on minnows on a black background, they were therefore in the dark phase. All injections were made intramuscularly, intra-peritoneal ones having no effect whatever. All the fish were killed fifteen minutes after the injections.
Doses of 0.05 mgm., 0.01 mgm., and 0.005 mgm. cause a contraction of the melanophores to take place over all the body. This pallor is very intense and frequently exceeds the average state on a white background. A dose of 0.001 mgm. causes paling which is more or less localised on the side of injection. The paling makes itself apparent first around the point of injection, and afterwards spreads over the side. 0.0005 mgm. only causes limited local pallor, while doses of 0.0001 mgm. and 0.00005 mgm. produce very local and transitory paling. 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 in
fig. 16. Where it is present, it is very intense and differs to no appreciable extent from the general pallor produced by the larger doses. There is no reason to suppose that the general and local effects are different in origin and may therefore be the same 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 pallor Intense pallor
n
Paling localised on side of injection
Paling locally round point of injection
Slight paling locally No change n t t Microscopic Results. Contracted Slightly stellate Local. General. Contracted » Stellate Normal » !» Normal » i t
The effect on the erythrophores is similar; contraction being produced. The xanthophores react in exactly the same way as the erythrophores. The end-points of both xantho-phores and erythroxantho-phores are difficult to state accurately as their reaction to black ground is neutral. It is therefore possible to obtain contracted chromatophores in sub-minimal doses. With these limits then it may be stated that the end-points were 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 same Ringer as that employed for the adrenalin experiments.
On a White Background. — The following doses were
administered: 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
region, it was found to be quite transitory and was probably the effect of moving and handling during injection. Microscopically there was not found to be any reason to suppose that " Infundin " produced any darkening (expansion) comparable to that stated by Abolin. In some cases the melanophores showed a tendency towards expansion, but this did not exceed what might be expected from individual differences. Moreover, there was not any agreement among these cases as to dosage.* The Ringer produced no effect on the colour.
The erythrophores and xanthophores were both expanded to a "stellate" or an "expanded" condition by these injections. The minimal dose is o.ooi c.c. (The normal phase is contracted.)
On Black Background.—The same range of doses was
employed. The only effect which was observed macroscopically was a paling in the dorsal region in the two largest doses. Microscopically this was shown to be confined to the lower layer of melanophores which were in the " stellate " condition although several cases showed even further contraction. Those injected with o.ooi c.c. showed slightly less contraction than this, and the lower doses produced no deviation from the normal condition on black background. The Ringer produced no effect. The erythrophores and xanthophores were consistently stellate where doses of o.oi c.c., 0.005 ac-> o r 0.001 c.c. were employed. Below 0.001 c.c. they varied between contracted and expanded—the normal reaction to a black background.
As Abolin had employed distilled water to make up the " Infundin" solutions the previous experiments are not repetitions of his work. Injections of 0.01 c.c. sterile extract made up in distilled water were, therefore, injected into pale minnows on a white background. The injections were made intramuscularly and intraperitoneally, but in neither case could any expansion of the melanophores be observed. The erythro-phores and xanthoerythro-phores gave the same reactions as were observed above when Ringer solution was employed. Injections of o. 1 c.c. of distilled water produced a slight irregular darkening when administered intramuscularly, but no effect was produced when injected intraperitoneally.
091 CC.
.
0905 n-.
0001,,
.
.
Contracted Sli
htly
etef~ate Stellate Stellate Stellate
Contracted Contracted Stellate
Stehte Sli
htly
stilate Stellate Slightly expanded
slif?
stete
Expanded Con
tracted
Contracted
Stellate
I
Expanded
Contracted Contracted Contracted Contracted
Slightly Expanded upanded Sli htly etcflate
Contracted Stellate
Contracted Sli
htly
st&te Sli
htly
ste%ate Stellate Slightly expanded Stellate Stellate Stellate
Sli
htly
steflate Sli
htly
stthe
Contracted
Lover
Layer.
Contracted Sli
htly
stetate Stellate Slightly stellate Sli
htly
steLe Expanded Sli
htly
st31ate Slightly .stellate
Contracted Contracted Sli
htly
steylate Stellate Sli
htly
ste'iate Stellate Stellate Stellate
No confirmation of Abolin's work was obtained therefore in these experiments. In fact, a reaction which resembled that obtained by Spaeth was observed. To test this further the following experiments were carried out.
Injections of Extract of Cod's Pituitary.—Extracts of fresh
cod's pituitary were made up in Ringer and injected, into dark minnows kept on a black background. The doses are given in grammes weight of the fresh gland (whole).
°-°°3 g"1-* o.ooi gm., and 0.0005 Sm- caused complete
pallor over all the body; and on microscopic examination the melanophores were found to be in the phases of "contracted" or " slightly stellate." The upper layer of melanophores, in the case of the dose of 0.0005 Sm-> w e r e> however, slightly less contracted, being in the phase of "stellate" for the most part.
0.0001 gm., 0.00005 gm>> a nd 0.00001 gm. all showed traces of local pallor, but this did not last long and disappeared before the fish were fixed so that microscopic examination only shows the normal reaction to black background.* The fish used in the 0.003 Sm' dosage were killed and fixed ten minutes after
injection. 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 hormone producing pallor in " Infundin," is illustrated by the following experiment.
One cod's pituitary was roughly f separated into its two lobes. Extracts were made up of approximately equal strengths of the two parts by weight. The following injections were made:—
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 that allowance for this source of error most be made as welL
.
.
.
.
.
.
Bars. Stripes Back
.
Bars. Stripes Back.
Sli
htly
steflate
Contracted Stellate
Fully
expanded Expanded Annulo- stellate
Folly
expanded Expanded
Full
expanid
Stellate Sli
htly
stekate Stellate Fully
expanded Stellate Slightly expanded Fully expanded Slightly expanded
Fult
mparr
ed
Stellate
Contracted Stellate
Fully
expanded Expanded Slightly expanded
Fully
expanded Expanded Expanded Expanded Sli
htly
stet
ate
Sli
htly
stekate Fully
expanded
Fully
expanded Expanded
Fully
expanded Expanded
Fully
expanded
A.
Contracted Contracted Contracted
Fully
expanded Stellate Annulo- stellate Stellate Sli
htly
stclfiate Annulo- stellate
B.
Sli
htly
steflate
Contracted Sli
htly
ste'fhte
Expanded Contracted Annulo- stellate
Fully
upand& Expanded Expanded
Sli
htly
steLte
Contracted Sli
htly
stetate
Expanded Stellate Slightly expanded
Fully
expanded Stellate Stellate Contracted Slightly stellate Contracted
Fully
" I nfundin" extracts were subjected to tests by peptic and tryptic digestion, and it was found that the hormone producing the foregoing results behaves in the same way as the one which acts on the melanophores of the frog. This points to a possible identity of the two hormones. If that is the case, then it constitutes another point in favour of the view that the melanophores of fish and amphibia are fundamentally different physiologically.
It may be stated then that in the minnow a hormone, present in the extract of posterior lobe of the pituitary, causes pallor and is possibly the same as that causing expansion of the melanophores 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 expansion is produced by posterior lobe extracts. The minimal dose for "Infundin" is about 0.001 c.c, while for an extract of cod's pituitary 0.0005 gm. of the fresh gland is the minimum.
7. Action of Pinealin and Thyroid.
The following injections were made with pinealin but, as will 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 injected
into two dark minnows on a black background. No change.
(5) 1 c.c. of a -rV gr. in 10 c.c. Ringer solution was injected
into two pale minnows on white background. No change.
Intraperitoneal Injection.
(c) 0.1 c.c. of a ^ gr. in 10 cc. Ringer solution was injected
into two dark minnows on a black background. No change.
(d) o.ic.c. ofa A gr. in 10c.c. Ringer solution was injected
into two pale minnows on a white background. No change.
Studies in Colour Changes of Fish
Similar injections were made with thyroid extract but they also were without effect. In all cases the same Ringer was used 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 and injected into pale and dark minnows. In the pale fish there was no change, but in the dark ones there occurred slight local pallor which was transitory.
Extracts of muscle and brain from a haddock were made up to a 5 per cent, solution and o.i c.c. injected into dark minnows. Apart from slight transitory local paling there was no change from the normal.
9- Conclusions and Summary.
The results obtained in this paper constitute a direct negation 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 enumerated agree with those of Spaeth. It was pointed out in the intro-duction that there may be differences between reactions induced by injections into the whole animal and those produced on isolated scales or portions of skin. The fact of identical reactions, however, suggests that in this case the method of reaction may be the same. This point has not been dealt with, as the reasons set forth below indicate that post-pituitary extract does not play any important part in the normal colour changes of the minnow.
The action of adrenalin appears to be peripheral whether injected or applied to isolated scales. The local and general reactions are merely due to differences in dosage. This being the case, the minimal dose, while being the smallest dose to produce contraction of the melanophores, is actually merely a gauge of concentration. The figure which is of importance is really that which gives the " minimal concentration " over the whole animal, producing a pallor comparable to that found in the normal reaction to a white background.
As regards the effects which the results of this research may have on the general theory of colour changes in fish, it must be pointed out that of the four hormones investigated, only two, namely adrenalin and post-pituitary extract, have any effect on colour. Furthermore, both these hormones produce a con-traction of the melanophores. Thus there are none of these four which produce expansion.
Whether adrenalin or post-pituitary extract produce the contraction in the normal conditions is the next problem. It does not seem probable, to the author, that post-pituitary extract could act in this way in the normal anomal. This would 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 is about ten times greater than that required to produce expansion in a frog, an animal a little more than ten times as large. Furthermore, the minimal dose of cod's pituitary for a minnow is 0.0005 Sm-> a nd 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 enough for paling two hundred minnows, assuming that the sensitivity of the melanophores and the concentration of the hormone in the gland are the same in both cod and minnow. Comparison of weights of the animals shows that the amount of hormone present would not have any effect in normal reactions.
Secondly, it will be observed that above the minimal dose required to produce contraction of the melanophores, expansion of the erythrophores occurs. Now the normal condition is that if the melanophores are contracted, then the erythrophores are also contracted. If, however, post-pituitary extract is the means of producing contraction of the melanophores, then expansion of the erythrophores would be expected to accompany it. So it will be seen that the artificial conditions do not coincide with the normal.
Any action, on the part of post-pituitary extract, on the erythrophores may also be doubted- For the action of this extract is to produce uniform expansion, and it has been shown that under the normal conditions of a black background ery-throphores in all phases may be seen in one and the same
individual. Further, such expansion is accompanied by con-traction of the melanophores, which does not occur on a black background. It must, therefore, be admitted that post-pituitary extract does not take part in the normal reactions of either the melanophores or erythrophores. The same applies to the xanthophores, which are identical in their reactions with the erythrophores.
It is not proposed here to deal with adrenalin further than has already been done. The reason for this may be briefly given. Adrenalin acts either on the nerve endings or on the 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 control of colour change, and it becomes impossible to deal with any one type of reaction without an extensive consideration of many other actions. This will, therefore, be dealt with at some further 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, 171.23a
Pouchet, 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 literature up 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.
FIG. 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 lateral line as indicated by the blood-vessel.
FIG. 12.—Photomicrograph to show the effect of "Infundin" injected into pale minnows on a white background. Compare with fig. 9.
FIG. 13.—Photomicrograph to show effect of an injection of an extract of cod's pituitary into a dark minnow on a black background. Compare with fig. 10. FIG. 14.—Photomicrograph to show effect of "Infundin" injected into a dark
minnow on a black background. Compare with fig. 10.
FIG. 15.—Photomicrograph to show the effect of adrenalin. This is the general action. Injected into a dark minnow on a black background.
FIG. 16.—Photomicrograph to shbw the effect of adrenalin. The effect here is localised. Injected into a dark minnow on a black background.
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