ABSTRACT
Records were made of the times required for the melanophores of the normal minnow to reach equilibrium when the fish is transferred from one to another of the following conditions : on an illuminated white background; on an illuminated black background; in darkness.
These times give further evidence of the parts played by nervous and hormonal mechanisms in the colour change of the minnow.
After section of the spinal cord between the 5th and the 12th vertebrae the fish darkens but gradually becomes pale again if kept on an illuminated white background.
Such fish can still show a slow colour change: dark on a black background, pale on a white background and intermediate in darkness.
Observations of the times required for these colour changes in the spinal minnow show that these no longer resemble those associated with the unoperated fish; rather, they resemble the time intervals associated with amphibian colour change.
Further consideration of the times required for colour change in the spinal minnow indicate that there is not only a hormone causing aggregation of the melanophores but also a hormone causing melanophore dispersion.
The part played by double innervation of the melanophores is considered.
I. INTRODUCTION
(a) Previous work
It is now well established that melanophores in teleosts are controlled by both nervous and humoral mechanisms. Sympathetic nervous control was shown to exist by Pouchet (1876) in the turbot and other flat fish. His results were confirmed by various workers but received their greatest extension through the work of von Frisch (1911). He showed that, in the minnow, fibres causing aggregation of the melanophores pass from a centre in the medulla along the spinal cord. At about the level of the 15th vertebra these fibres enter the sympathetic chain and there run forwards and backwards, passing out with the spinal nerves to supply the melano-phores of the body and with a branch of the trigeminal nerve to supply those of the head. Von Frisch found that when he cut through the fibres of this system the region of the body separated from the centre in the brain quickly became dark and could no longer take part in the normal colour changes of the fish. However, he observed that when such a fish was kept for some time upon a white background the denervated region gradually became pale; if the animal was then placed on a black background the denervated region slowly darkened again. At that time he did not pursue this matter further. Later experiments by various workers showed that melanophores could be affected by chemical substances, and Hogben and his co-workers demonstrated the role of hormones in the colour changes of Amphibia. This knowledge suggested that similar mechanisms might play a part in the colour changes of fish. That they do so has been shown by a number of workers on elasmobranchs (Lundstrom & Bard, 1932; Hogben, 1936; Parker, 1938; Waring, 1938) and teleosts (Parker, 1934; Abramowitz, 1936, 1937; Osborn, 1938; Hogben & Landgrebe, 1940; Neill, 1940; Healey, 1940; Vilter, 1941).
Smith (1931) continued von Frisch’s observations on the minnow. He denervated head melanophores by cutting through the trigeminus and body melanophores by cutting through spinal nerves. He was also able to observe a slow colour change in the denervated region and showed that this depended upon an intact blood circulation. He concluded that some hormone in the blood caused the aggregation of the melanophores and suggested that it might be adrenalin. On the other hand, Parker and his school have shown that denervated areas resulting from small transverse cuts in the tails of Fundulus heteroclitus and Ameiurus nebulosos still show some colour changes and have maintained that these are caused chiefly by neurohumours which diffuse into the denervated region from the endings of the neighbouring intact nerve fibres. The author (1940) observed the reactions of melanophores in the minnow after the spinal cord had been cut anterior to the 15th vertebra. According to von Frisch (1911) the entire peripheral chromatic nervous system should be put out of action by such an operation (as far as connexion with the brain is concerned) and the possibility of neurohumours from nerve endings playing a part should not arise. Observation showed that the fish still change their colour slowly on a white or black background, and that this colour change depends upon the presence of the pituitary gland. The latter produces a hormone which is responsible for the paling of the operated fish on a white background. There were strong indications that it may also produce a hormone which causes darkening of the fish on a black background; but the evidence for this was not conclusive.
It has been reported many times that section of chromatic nerve fibres in teleosts results in the darkening of the denervated region. Some workers have attributed this darkening to the paralysis of sympathetic aggregating fibres and others to a stimulation through cutting of parasympathetic dispersing fibres. Since the presence of aggregating fibres is not disputed there remains the possibility of a double innervation of the melanophores : a sympathetic innervation causing aggregation and a parasympathetic innervation causing dispersion. Parker has fully presented his case in favour of this view (1948). In the case of the minnow, Giersberg (1931) showed a darkening after the injection of certain parasympathetic excitants. He also found that after treatment with ergotamine followed by acetylcholine he could produce melanophore dispersion by electrical stimulation. Von Gelei (1942) carried out further experiments along these lines. After treating minnows with ergotamine and acetylcholine he cut the sympathetic chain and the spinal cord at different levels before applying electrical stimulation to the medulla. His results indicated that the fibres responsible for melanophore dispersion do not follow the same path as those responsible for melanophore aggregation as found by von Frisch (1911) but pass to the sympathetic chain from the spinal cord by the first or second spinal nerve. However, he concluded that these dispersing fibres play no significant part in the colour change of the normal animal. It is still possible that they may play a part in the colour change of minnows whose spinal cords have been cut posterior to the 2nd spinal nerve (von Frisch, private communication).
(b) Present work
In a number of papers Hogben has drawn attention to the importance of time relations in the study of chromatic responses and, from a consideration of them, he and his co-workers have arrived at a number of significant conclusions (Hogben, 1943; Neill, 1940). In the author’s earlier experiments (1940) the colour changes of the minnow were estimated macroscopically by gross colour matching against standards. All fish were tested on different backgrounds before use to ensure that they showed the same tint under similar conditions, and those which did not conform (through different sizes or concentrations of melanophores or through disease) were discarded. Nevertheless, for the timing of colour changes the method of estimating the degree of dispersion of the melanophores by the use of the melanophore index (Hogben & Slome, 1931) offers great advantages and was therefore adopted in the present work. Fig. 1 shows the various degrees of dispersion which are represented by the M.I. values of 1-5. The minnow has both epidermal and dermal melanophores distributed on the dorsal and dorso-lateral surfaces of the body and extending to some extent into the fins. These regions are fairly sharply demarcated from the ventro-lateral and ventral surfaces on which there are few if any melanophores. The melanophores themselves are of two sizes. Large epidermal and dermal melanophores are associated in groups which macroscopically form a variably well-defined pattern. This takes the form of a thin mid-dorsal line, scattered dark flecks on the dorsal and dorso-lateral surfaces and a wide dark stripe along each side. This dark stripe will be referred to as the ‘lateral stripe’. Below it the region of few or no melanophores begins. Fig. 2 shows diagrammatically the posterior region of a minnow with the main distribution of melanophores. Those parts of the dorsal and dorso-lateral surfaces not occupied by these large melanophores are provided with very much smaller melanophores in both the epidermis and the dermis. At the root of the tail fin the lateral stripe usually becomes somewhat enlarged. This is the region A in Fig. 2. The region of small melanophores dorsal to the region A is referred to as region B. Readings of the M.I. were taken at B in the case of unoperated fish, but in the case of operated fish readings were also taken at A and often also on the dorsal surface (small melanophores) and the lateral stripe (large melanophores).
(c) Source and general treatment of fish
Minnows were obtained from four sources. In earlier experiments carried out in 1940 the fish came from the River Dee at Aberdeen and from the River Tweed. They were kept in the laboratory in stock tanks with a bottom of brown pebbles, supplied with running water and fed on raw finely minced meat and Cura’s ‘XL’ fish food. In later experiments carried out at Aberystwyth in 1949 the fish were taken from the River Dovey and from Fron Gogh Pool near Trisant in Cardiganshire. These fish were kept in slate stock tanks and in light stone sinks with slate bottoms. Aberystwyth tap water, if allowed to stand, readily dissolves lead from pipes. For this reason the water was first run fast for 20 min. to ensure an uncontaminated supply from the mains. It was then collected in the tanks and treated with chalk and a rapid stream of air for 30 min. in order to reduce acidity and to remove chlorine. After these precautions had been taken the minnows were able to survive in it. They were fed upon finely minced meat and dried food supplied by Messrs Haig.
It has been shown (Healey, 1940) that minnows from different sources may differ in some of their colour change reactions. In this case no essential differences were observed in the minnows from the various sources, as far as they were studied.
Before using them for experiments fish were kept for at least a week in small glass aquaria with slate bottoms to accustom them to more restricted space and to the movements of an observer. Excitement and its consequent pallor (von Frisch, 1911; Healey, 1940) were thus largely avoided. During experiments they were usually fed little if at all in order to avoid fouling the water.
All the experiments were made in a dark room with standard illumination, namely, a 40 W. domestic electric lamp at 1 m. from the fish. The temperature of the water was maintained throughout at 12 ±0.2° C., unless otherwise stated.
II. THE COLOUR CHANGE OF NORMAL MINNOWS
(a) General experimental procedure
The minnows used in this series of experiments were about 3 cm. long. The containers were 250 ml. conical flasks with several holes blown in the bottom to allow rapid drainage while preventing the escape of the fish. Six of these flasks, each containing one fish, stood on glass props in 4 cm. of water in a glass aquarium. This rested upon a black or white painted metal tray with sides 7 cm. high. The tray and aquarium were immersed in a thermostatically controlled bath. A stream of air was passed through the water in each flask and through the water in the aquarium. In this way there was adequate aeration and thorough mixing of water. Since it was difficult to remove the aerator tubes quickly from the flasks without disturbing the fish, they were cautiously taken out 20 min. before the experiment began. For the remainder of the time the fish obtained their oxygen in adequate supply from the water in the flasks and in the aquarium.
The fish were first kept for 7 days in white or black painted aquaria whose temperature was not controlled, depending upon the background adaptation required at the beginning of the experiment. The level of water in these aquaria was then carefully reduced until the fish could easily be made to swim into the flasks, i.e. all handling of the fish was avoided. After 12 hr. in the flasks on the appropriate background, arranged as described above, the experiment proper began.
When timing the colour change from white to black and from black to white backgrounds the whole aquarium with its contained flasks was quickly lifted on to a tray of the appropriate colour which was already lying in the constant-temperature bath beside it. The fish were then removed from the flasks after various time intervals and observed. This was done by lifting the flask from the aquarium (and so draining it almost as quickly as it could be removed) and tipping the fish, which almost always came out head first, on to a grid made of glass rods cemented to a glass plate. This grid method has been fully described by Neill (1940). The fish was quickly covered with another glass plate and observed under the microscope. With practice the time elapsing between the initial removal of the flask from the aquarium and the observation of the melanophores did not exceed 6 sec.
When timing colour change from black or white backgrounds to darkness, i.e. taking observations in the dark, a closed box which could be very dimly illuminated from the inside and had a small aperture on the top was covered by a glass plate and used as a support for the grid. At the moment of taking out the flask from the aquarium the box light was switched on so that the position of the fish on the grid could be seen. As soon as the fish was covered the box light was switched off and the fish was examined under a microscope with concealed substage fighting. This extra procedure added about 2 sec. to the time elapsing between removal of the flask and observation of the melanophores.
When timing colour change from darkness to black or white backgrounds the fish were kept in their flasks on trays of the appropriate colour in darkness for 10 days before the fight was switched on. The water in the aquarium (and therefore in the flasks) was replaced by means of cautious siphoning every 2 days in order to avoid pollution.
Hogben & Landgrebe (1940) have shown that in the case of Gasterosteos records of M.I. values cannot be made from consecutive readings of the same fish, since readings are no longer accurate after the fish has been handled. It therefore seemed advisable to avoid repeated handling of the minnows and to discard a fish after one reading had been taken. Accordingly, each fish was only used once.
Observations of melanophores in unoperated fish were restricted to dermal melanophores in the region already referred to as B (§ 1 (b)). When a minnow is removed from its flask and observed as described above its melanophores change their form very rapidly, so that it is impossible to make any reasonable estimate of their degree of dispersion in more than one region at a time. Attempts to fix the melanophores by plunging the fish into hot fixatives did not meet with success (Hogben, 1943; Parker, 1943).
The M.I. readings were plotted graphically, each point on the graph representing the mean M.I. value of a group of at least ten fish. The limits of a vertical line drawn through each point show the lowest and highest M.I. values found within the group.
(b) Observations
The results presented in this section agree closely with those obtained by Hogben & Landgrebe (1940) with Gasterosteos and by Neill (1940) with some other teleosts, and the treatment given by these workers may be applied here.
In the period between the presentation of a visual background stimulus and the response of the melanophores three components play a part:
(a) The time required for the eye to react to the stimulus.
(b) The time required for transmission through the co-ordinating system.
(c) The reaction time of the melanophores.
(a) is only a few seconds. If there is only nervous co-ordination (b) is only short and is measurable in seconds. As far as nervous control is concerned (c) is also short; e.g if a decapitated and eviscerated minnow whose melanophores have reached their maximum degree of dispersion is stimulated electrically on the bare surface of its spinal cord the melanophores show a M.I. change from 4·6 to 1·4 in 20-30 sec. After stopping the stimulus the melanophores return to their original state in little more than 1 min. On the other hand, the melanophores of the minnow only react relatively slowly to hormones, requiring about 2 hr. for a maximum response (Healey, 1940).
Fig. 3 shows the responses of the melanophores when the minnow, fully adapted to an illuminated black background, is transferred to an illuminated white background, and vice versa. There is a rapid colour change for the first few minutes, i.e. its rate is comparable with that which we should expect in the case of nervous co-ordination. At the end of 15 min. this rate has become greatly reduced and the colour change continues slowly, only becoming complete after about 2 hr. Other methods (Healey, 1940) have already shown that in the minnow there is not only nervous co-ordination of colour change but also chemical co-ordination through the mediation of at least one hormone. The effects of this double system may be well seen here: the initial rapid colour change is the result of nervous co-ordination, while the slow colour change which follows it is the result of the action of a hormone.
When the animal is fully adapted to an illuminated white background the mean M.I. value is 1·25. When black-adapted the value is 4·8. Different individuals agree fairly closely.
Fig. 4 shows the responses of the melanophores when the minnow is transferred from equilibrium on black and white illuminated backgrounds to darkness. Equilibrium in darkness is only reached after about 45 hr., a very much longer time than that required for illuminated background equilibrium (Fig. 3). This shows once again (Hogben & Landgrebe, 1940; Neill, 1940) that in teleosts the mechanisms which are responsible for the colour changes following transition from illuminated backgrounds to darkness are different from those following a change of illuminated background. In the latter case the nervous system controls the rapid colour change and is reinforced by hormonal control; but the slow colour change in darkness is the result of a gradual change in hormone concentration, the nervous system here playing no part.
Fig. 5 shows the responses of the melanophores when the minnow is transferred from equilibrium in darkness to black or white illuminated backgrounds. Here the nervous system again plays a part, and the times are of the same order as those shown in Fig. 3.
The minnow, like many other animals showing colour change (Hogben, 1943), reacts not only to the tint of the background (secondary response) but also to the intensity of illumination (primary response). In some blinded fish observed macroscopically this primary response is evident within a few seconds of transference from darkness to light; in others it is difficult to detect in this way (Healey, 1940). The minnow when adapted to darkness has a mean M.I. value of 2·1. The blinded minnow under standard illumination has a mean M.I. value of 3·0 after 4 days; but this becomes progressively higher with time, so that it is not possible to use the value for a trustworthy estimate of the extent of the primary response, it can only serve as some indication. The curves presented in this section do not show the presence of this primary response. The reason may lie in the technique adopted together with the fact that the degree of this response varies considerably among different minnows; but probably the technique is the more responsible. The minnow changes colour with extreme rapidity when handled, and even the few seconds required to read the M.I. may be-sufficient to render the results suspect as far as absolute values are concerned, while allowing them sufficient accuracy to show the general trends. A different technique may well throw light on this point.
III. THE COLOUR CHANGE OF MINNOWS AFTER SPINAL SECTION BETWEEN VERTEBRAE 5 AND 12
(a) General experimental procedure
The minnows used in this series of experiments were about 5 cm. long. They were anaesthetized in 0·5% urethane solution and then supported with filter-paper and rubber bands on a piece of grooved paraffin wax in a small tray provided with inlet and outlet tubes. From the former the fish was supplied with a stream of 0·25 % urethane which could be temporarily replaced by water if the respiratory movements of the animal became weak. The operation was carried out under a bar binocular with twin lighting, visibility being further assisted by continuous washing of the operation site with a stream of Ringer solution. After sewing up the wound the fish was weighted and suspended with its number ticket in an aquarium painted black or white. The procedure has already been fully described (Healey, 1940). The aquaria were placed in the constant temperature tank and illuminated as already described (§ I (c)). At noted intervals the fish were lifted out and examined in a container made of plasticine and glass in such a way that the head of the fish rested in a hollow filled with water, while the tail lay over a glass plate and could be examined through the microscope. The fish usually lay quite still. When working in fight the first reading could be made within 6 sec. from the time when the fish was first lifted; in darkness the time required was within 8 sec.
The M.I. was read in the same region (B) as in the case of the unoperated fish, but in many cases, since the colour change is now much slower, additional readings were taken in region A, in the dorsal surface and in the lateral stripe (§ I (b)). In such cases the first position was read again to make sure that the M.I. value had not changed in the time (within 22 sec.). Some results obtained from different regions are presented in § III (b). After the M.I. value had been read the fish was replaced in its aquarium.
In making these observations some precautions were taken in an attempt to lessen the danger of subjective influences on the part of the observer. The fish were removed from their aquaria in different order at different times; they were frequently rearranged in the aquaria; the reference number labels were only looked at after the reading had been taken. As a result the observer had no remembrance of a previous reading of an individual fish and, therefore, expected no particular M.I. value. Such a precaution would hardly have been necessary if all the melanophores in the field of view had shown the same degree of dispersion; but in many cases this was not so, and an estimate had to be made from the general appearance.
It will be noted that in this series of experiments the fish were not discarded after a reading had been taken but were replaced in their aquaria and read repeatedly. In estimating the likelihood of such repeated readings affecting the accuracy of the results the following points deserve consideration :
(i) Unlike the unoperated fish these spinal animals showed very’ wide variations in M.I. among one another when under the same experimental conditions (cf. figures showing responses of operated and unoperated fish). Consequently, readings derived from different groups of fish gave very irregular graphs whose tendencies could not be well determined. This is a point of some consequence when we bear in mind that the whole object was a determination of time relations as accurately as possible.
(ii) Groups of fish read four times a day gave the same type of curve as did other groups read only once daily. The actual values obtained differed, but such differences could well be attributed merely to the wide variations between individual fish ((i) above).
(iii) Attempts to repeat a series of observations on the same group of fish in a different way (e.g. black to white with frequent observations followed by white to black and then black to white again with infrequent observations) showed that the time relations were of the same order, but that the actual M.I. values were somewhat different as a result of degenerative changes which often appear in the melanophores some time after spinal section.
(iv) Readings were taken of some fish which were then kept for a longer time than usual in the plasticine container. When it was obvious that they had become paler they were returned to their aquarium. Another reading within 2 hr. gave results which agreed well with the other observations, both when a fish was in the process of changing its colour and after it had reached a steady M.I. value.
(v) An experiment was made in which the colour change of a group of fish was read at intervals in the usual way but with this addition : between each reading and not less than 3 hr. before the next reading the fish were lightly pinched with forceps or gently tapped with a glass rod. As a result they became excited and went very much paler (von Frisch, 1911; Smith 1931). In spite of this the readings gave a graph which agreed with the type obtained when no mechanical stimulation was applied.
(vi) One might say that, in any case, the fish had already been most severely ‘handled’ in the course of the operation and subsequent suspension in aquaria.
In short, unless each observation had been made on such a large group of fish that the individual differences could have been neglected, nothing would have been achieved by using separate groups.
When the observations on a fish were completed the animal was killed by immersing it for 6 sec. in boiling water. After being suspended for 2 weeks in a formol-acetic acid mixture to harden and decalcify it, it was divided by a median vertical longi-tudinal cut and examined under a low-power microscope. In all the fish included in this paper the site of the spinal section was found to lie between the 5th and 12th vertebrae.
(b) Responses of melanophores from the time of the operation to equilibrium on illuminated black and white backgrounds
The fish taken from the stock tanks had a M.I. of about 3·2. The urethane caused some slight additional darkening, but almost immediately after the cut in the spinal cord had been made the fish became still darker in colour. This darkening continued to increase after the fish had been put on to a black or white background and reached an individual maximum within 30 min. The fish had been back in water for about 25 of these minutes, a time in excess of that required by an unoperated animal to recover from the darkening effects of urethane anaesthesia, i.e. the darkening results from the operation. Mention has already been made (§ 1(a)) of this melanophore dispersion which follows section of chromatic fibres. Further attention will be given to it in § III (f).
At noted times after the operation the M.I. values of the fish were read. Fig. 6 shows these readings expressed graphically. Curve (a), representing the responses of the melanophores when the fish were placed on an illuminated black background after the operation, shows two things : first, from the mean M.I. values, that the dispersion of the melanophores following the operation becomes less intense and reaches a steady condition after 4-5 days; secondly, that there, s a considerable difference between individuals. Curve (6) shows the response when the fish were placed on an illuminated white background after the operation. In this case the M.I. becomes much lower and finally reaches a steady value after 8-9 days. The differences between individual fish on the M.I; scale are very marked. In this connexion it may be emphasized again that the vertical lines in the figures only join the highest and lowest M.I. readings. In fact, for curve b (Fig. 6) at 9 days only three fish had M.I. values above the mean, namely, 2·0, 2·6 and 3·6. The other nine fish contributing to this curve were below the mean, i.e. the chance presence of even one divergent fish in a group can give a long vertical line in this type of representation. Nevertheless, the presence of divergent fish within a group was so common that there was no question of ignoring it and treating such fish as exceptions. Indeed, in most cases the majority of the fish within a group did not conform so nearly to the mean as in the example quoted here.
In the case of the operated fish, as already mentioned, not only was the region B (§ I (b)) read but often also the region A and sometimes the dorsal surface and lateral stripe. Fig. 7 shows the reactions of the melanophores from these different regions in two fish nos. 72 and 78, both of which contributed to Fig. 6. In both cases the plots of the various melanophore regions in an individual all lie reasonably close together. Since similar results were obtained from other fish, it may be concluded that the plots of region B give a fair indication of the behaviour of the melanophores in other regions of these spinal fish. The great difference in the reactions of individual fish has already been pointed out. It is clearly demonstrated in Fig. 7. Fishes nos. 72 and 78 both had a M.I. of 5·0 immediately after the operation, but on an illuminated white background they paled at very different rates and to a very different extent; no. 72 reached a M.I. value of 1-2 in about 30 hr. and subsequently became only a little paler; the M.I. value of no. 78 fell fairly regularly to reach a level of 3-4 after 8-9 days.
(c) Responses of melanophores when spinal fish, equilibrated to an illuminated background, are subjected to background reversal
Operated minnows which had been allowed to reach a steady M.I. value by keeping them for 9 days after the operation in black and white illuminated aquaria were subjected to a reversal of the background to which they had become equilibrated. Fig. 8 shows the results of such an experiment. Considering first the background change from black to white, shown in curve (a): the group of fish had reached a mean M.I. value of 4·9 after the preliminary period of 9 days on a black background after the operation. In Fig. 6 the group of fish left on a black background showed a lower M.I. value after the same time. But it has been pointed out that there is considerable variation; in the present group (Fig. 8) the mean remained high. After placing the fish on a white background this initial M.I. value fell to 3·0 in 5 hr., to 1·5 in 48 hr. and to 1·25 after another 48 hr. The curve 8(a) also shows that the later M.I. values are all much closer together than they are in the case of the white-equilibrated fish in Fig. 6(6). This would appear to be nothing more than a matter of chance; the final variation in this group happened to be slight.
Considering, secondly, the background change from white to black, shown in curve 8(b): the group of fish had reached a mean M.I. value of 1·7 after the preliminary period of 9 days on a white background. When the group was placed on a black background the mean M.I. value rose to 2·6 in 5 hr. and to 4·25 after about 50 hr. After this it remained at a steady level.
The two curves in Fig. 8 show time relations of a different order from those obtained from unoperated minnows (Fig. 3). They resemble the time curves associated with amphibian colour change (Hogben & Slome, 1931). The reaction time to pituitary hormones of the melanophores of the minnow under these conditions of operation is of the order of 90 min. (Healey, 1940) and the eye is still the receptor. The long time required for colour change is therefore consistent with the hormonal control existing in this fish.
The final M.I. values indicated by curves 8(a) and (b) are not the same as the initial values of 8(b) and (a). This might be attributed to the differences which have already been seen to exist between individuals.
(d) Responses of melanophores in operated minnows equilibrated to illuminated black and white backgrounds on transference from light to darkness and the reverse
The primary response is more obvious in the operated than in the normal minnow, since in the former it is not rapidly masked by interference from the nervous system resulting from visual background stimuli. The operated minnow equilibrated in darkness has a M.I. value around 3·6. (Again there is considerable variation.) The blinded spinal minnow in fight gradually becomes darker, the depth of colour increasing for many days, so that it gives us no reliable information about the extent of the primary response. However, the spinal but seeing fish equilibrated to darkness shows the primary response quite clearly when it is exposed to light; the change in mean M.I. value is from about 3·6 to about 3·8 in Fig. 10. Similarly, the spinal illuminated fish shows a relatively rapid initial paling when transferred from light to darkness; from about 4·6 to 4·2 and 2·4 to 1·9 in Fig. 9. This response requires about 6 min. for its approximate completion. It appears to be largely, if not entirely, the result of local stimulation of the melanophores (Healey, 1940).
Fig. 9 shows the responses of the melanophores of spinal minnows which have been equilibrated to an illuminated black or white background and then transferred to darkness. The fish from a black background show a fairly rapid fall in the M.I. value as a result of the primary response. Thereafter the fall continues slowly and reaches a steady value of 3-6 after about 100 hr. The fish from a white background, after showing the initial fall in M.I., gradually become darker, reaching a steady value after about 160 hr. Here, as in the similar case in § 11(6), the hormone secretion which supplements rapid nervous action in the intact minnow is no longer taking place through stimulation of the eyes. Its slow change in concentration results in the new equilibrium level for darkness.
When such fish, equilibrated to darkness, are transferred to illuminated black and white backgrounds, the establishment of new levels of hormone concentration in response to the optic stimuli results in the attainment of new equilibria, as shown in Fig. 10. These are not the same fish as those used for Fig. 9, the initial values of the two groups being here quite close to one another (M.I. 3·6 and 3·7). Very soon after the Light is switched on the primary response is seen and the mean M.I. rises (to 3·8 and 3·9). The fish on a black background then darken slowly to reach a steady value after about 120 hr. The fish on a white background become paler and reach a steady value in about the same time.
(e) A consideration of the responses of the melanophores with regard to the bi-humoral hypothesis
These results obtained with spinal minnows increase the existing knowledge of hormonal control in this fish (Healey, 1940) and agree with results obtained with other teleosts by Hogben and his co-workers (Hogben, 1943). According to Hogben’s hypothesis (i) the colour changes resulting from the transition from light to darkness and vice versa are the result of excretion or secretion of the B (melanophore dispersing) hormone from the pars intermedia of the pituitary gland; and (ii) those following transference from black to white illuminated backgrounds and vice versa are the result of secretion or excretion of W (melanophore aggregating) hormone or of direct nervous control ‘secondarily superimposed upon, and to a greater or less extent replacing, a more archaic humoral mechanism’ (Hogben & Landgrebe, 1940). This hypothesis is supported by the results obtained from the timing of colour changes in Amphibia (Hogben & Slome, 1936), elasmobranchs (Waring, 1938) and the eel (Neill, 1940), the relevant times being those required for the change
The presence of a W hormone causing melanophore aggregation in the minnow has already been demonstrated (Healey, 1940), but the existence of a hormone which causes melanophore dispersion in this fish, although strongly indicated, needs further confirmation. The colour changes of the spinal fish which have been described in this section do not indicate that any active nervous response to background tint is playing a part in them. Nevertheless, the work of von Gelei (1942) suggests that dispersing fibres which have not been cut by the spinal operation may still be influencing the melanophores : that is, there exists the possibility, not precluded by the evidence at hand, that these fibres may at least be responsible for some slight melanophore dispersing influence which is either (a) constant under all conditions of illumination or (b) greatest in light and least in darkness. These possibilities require further investigation but do not concern the present argument. The order of time required for the colour changes under consideration can only be associated with hormonal control. Following Hogben and his co-workers we may argue thus: If there is only the W hormone playing a part its presence in varying amounts in the blood will be responsible for the colour changes observed, namely, pale on an illuminated white background (high concentration of W), intermediate in darkness (medium concentration of W), dark on an illuminated black background (low concentration of W). Therefore transition from equilibrium on a black background to equilibrium on a white background should involve the liberation of more W hormone than transition from a black background to darkness or the transition from darkness to a white background, i.e. b.Tw should be greater than bTd and dTw. But the figures given above show that this is not so. Again, the transition from equili-brium on a white background to equilibrium on a black background should involve the elimination of more W hormone than transition from a white background to darkness or the transition from darkness to a black background, i.e. wTb should be greater than wTd and dTb. Again the figures show that this is not the case. By this argument, therefore, the colour changes observed in the operated minnow cannot be due to a single W hormone. We therefore have a further indication of the presence of another hormone, B, antagonistic to the W hormone and causing melanophore dispersion in the minnow. The different rates of accumulation and elimination of these two competing hormones can then be made to account for the various times elapsing between establishment of different equilibrium conditions.
(f) A consideration of the responses of the melanophores with regard to their double innervation
In § 1(a) reference has been made to some observations which indicate that melanophores are supplied with both aggregating and dispersing nerve fibres. Parker and his school maintain that the cutting of chromatic nerve fibres causes darkening through stimulation of dispersing fibres, i.e. the darkening which follows the operation of spinal section, as described in the present paper, might be interpreted as being a result of such stimulation. On the other hand, according to von Gelei (1942), dispersing fibres in the minnow pass out of the spinal cord well before the level of the cuts made in this series of experiments. There is thus lack of agreement on this point, and further work is necessary to clarify it. However, some of the results obtained here do merit mention in this connexion.
(i) A comparison of Figs. 6(b) and 8(a) shows that, although the initial M.I. values are about the same in both, the rate of paling is much greater in the latter. Protago-nists of the theory that cutting stimulates dispersing fibres might say that this is another example to prove their point. They might perhaps add that von Gelei’s description of the track of the dispersing fibres cannot be complete; that there are, in addition, other dispersing fibres which follow the same path as those causing aggregation and are therefore cut by the present spinal section; that after some time the stimulating effect of the cut decreases and the melanophores are now affected only by the hormonal constitution of the blood and by von Gelei’s dispersing fibres which, according to him, do not seem to play a very active part. Antagonists of the theory of stimulation through cutting might say, as readily, that the paralysis of aggregating fibres is not the only result of spinal section. The severing of their nervous connexions with the brain may cause considerable physiological changes in the melanophores which may well affect their reactivity to hormones; also, spinal section is, at the best, a violent and undiscriminating operation.
(ii) It is further of interest in this connexion to compare the rates of paling on an illuminated white background of spinal fish which have received different preliminary treatment (Healey, 1940). The fish represented in Fig. 6 were kept in the stock tanks before their spinal cords were cut. The curves shown in Fig. 11 were obtained thus: two groups of unoperated fish, (a) and (b), were placed respectively on black and white illuminated backgrounds. At the same time another group (c) was subjectedto spinal section and placed on a black background. After 9 days groups (a) and (b) were also operated and all three groups were then placed together on a white back-ground. Their M.I. values were then read and plotted to give Fig. 11. In this experiment it was not convenient to use the thermostatically controlled tank, and the temperature accordingly varied between 13·5 and 15·0 ° C.; but this variation applied equally to all three groups so that the results are comparable with one another if not with Figs. 6 and 8(12 ° C.). We see that in groups (a) and (b) the previous adaptation to black and white backgrounds made no difference to the effect of spinal section excepting that group (a) was rather darker than group (b) immediately after the operation and for the next 30 hr. This first difference in colour may be attributed to the very different concentrations of hormones in the blood of the two groups at the time of the operation. After 30 hr. on a white background this difference had become much reduced. Otherwise, in spite of the previous black and white adaptation, there was no essential difference between the rates of paling of the two groups.
Group (c) became paler more quickly than did groups (a) and (b), a result which agrees with the comparison already made above between Figs. 6 (i) and 8(a). One may draw the conclusion that this first paling immediately after the operation is a result of processes which are not identical with those responsible for paling which may occur later, i.e. the operation of cutting the spinal cord would appear to initiate some process which, for a time, actively combats the paling of the fish.
(iii) The slow changes in hormone concentration which result when the unoperated minnow is transferred from illuminated black or white backgrounds to darkness are seen in Fig. 4; the M.I. value at equilibrium in darkness is about 2·1. Fig. 9 shows the chromatic behaviour of the spinal minnow under similar conditions; here the M.i. reaches a steady value of about 3·6. Further, the condition of darkness equilibrium in the case of the spinal minnow is only reached after a much longer time than that required by the unoperated minnow. These differences in behaviour of normal and spinal minnows might possibly be claimed as evidence for double innervation; thus the suggestion might be made that in the unoperated fish in darkness the parts of the nervous system responsible for aggregation and dispersion of the melanophores might be competing weakly for control with the former dominant. Then, although the hormone concentrations at the end of 45 hr. are not in darkness equilibrium, the nerves alone might be responsible for the steady state of the melanophores as shown by the present technique. Secondly, in the spinal fish only the dispersing system indicated by von Gelei is playing an active part (at least, so far as connexions with centres in the brain are concerned). Acting against this, in the case of the spinal fish transferred from equilibrium on an illuminated white background to darkness, is a considerable concentration of W hormone. As the latter is gradually eliminated the M.I. value rises but takes longer to reach equilibrium, as it now has to pass through a greater range. Similar arguments might be advanced to account for the chromatic behaviour of the spinal fish transferred from an illuminated black background to darkness. On the other hand, those who are not convinced that dispersing fibres play an active part might say that aggregating fibres maintain a certain slight tonic effect and produce a darkness equilibrium at a fairly low M.I. value in the unoperated fish; in the operated fish, transferred from an illuminated white background to darkness, this effect is no longer exerted, and the W hormone concentration has to fall much further so that there is a longer time required to reach the steady state. Finally, one might express the view that the nervous system may play no part in darkness equilibrium in either the unoperated or the operated fish, but that cutting the spinal cord may produce physiological changes in the melanophores themselves which profoundly affect their reactions. In short, the interpretation of these results is uncertain and further experimental work is necessary.
SUMMARY
The work described here was carried out in the Department of Natural History, Aberdeen University, with the help of a grant from the Rockefeller Foundation. This is gratefully acknowledged. The author’s thanks are due to Prof. Lancelot Hogben for administering this grant for his benefit and also to Mr R. M. Neill for his readiness to provide facilities in Prof. Hogben’s absence. Further experiments were carried out at Aberystwyth. These were made possible by the facilities provided by Prof. T. A. Stephenson and to him also the author wishes to record his thanks.