The ammocoete of Lampetra planeri (Bloch) contains a single carotenoid, lutein, partly esterified and partly in the free state. The pigment is distributed in lipophores in the sub-dermal tissues, and plays no part in the colour changes of the animal.

The epidermis of the tail contains numerous sensory cells of a type previously undescribed, which satisfy the requirements predicted for the photoreceptors responsible for the light reaction of the animal.

Evidence is presented that these sensory cells are innervated by branches of the lateral line nerves.

Similarities are indicated between the sensory cells of the ammocoete and simple photoreceptor systems of certain invertebrates.

This paper describes investigations on the nature, distribution, and innervation of the photoreceptors in the skin of the ammocoete of the brook lamprey, Lampetra planeri (Bloch). Although it has been known for many years that the skin of lampreys and their ammocoete larvae is sensitive to light, the photoreceptor cells concerned have never been identified. Young (1935) showed that the tail is the most sensitive region of the body, and also that the receptors are connected with the central nervous system by way of the lateral line nerves. My own recent analysis (Steven, 1950) of the principal physiological characteristics of the light reaction of the ammocoete has shown that it resembles in many respects those of invertebrates, such as Mya and Ciona, which lack image-forming eyes. With the exception of the cave-dwelling urodele, Proteus anguineus (Hawes, 1946), the light reaction of the lamprey is the only example of a simple photoresponse in a vertebrate of which we possess any precise knowledge. We are at present almost completely ignorant of the photosensitive processes of animals at levels of organization below that of the image-forming eye, and a detailed study of these simpler systems offers one of the most promising approaches to an understanding of the pattern of activity common to all ‘visual’ processes.

Two principal lines of investigation have been followed in this work:

1. A study of the carotenoid pigments of the ammocoete, and their histological distribution.

2. A study by neuro-histological techniques of the sensory cells of the skin and their innervation.

Ammocoetes of L. planeri contain two types of pigment, melanins and carotenoids. The former are contained in melanophores of a typical fish type, which expand and contract under different conditions of illumination and are responsible for the colour changes of the animal. They are distributed principally in the sub-dermal tissue around the myotomes and spinal cord, but are absent from the outer parts of the dorsal and ventral lobes of the tail fin. They were not considered to be of any interest in connexion with the light reaction. No description appears to exist of the carotenoid-containing cells, and since pigments of this class are closely associated with photoreceptors in a wide variety of organisms, a study of their distribution was clearly relevant to the problem.

The carotenoids of the skin were investigated by procedures similar to those described by Zechmeister and Cholnoky (1941). Details of the method used are given by Steven (1948). Portions of the skin of the tail and body regions of several large ammocoetes were separated carefully from the underlying muscle and the viscera. The combined portions were weighed, chopped with scissors and ground with fine sand mixed with anhydrous sodium sulphate. The finely ground mixture was then extracted exhaustively with light petroleum ether (B.P. 40°-60° C.) containing 2 per cent, of pure methyl alcohol. The combined extracts were transferred to pure petroleum ether by diluting with water, dried by shaking with anhydrous sodium sulphate, filtered, and evaporated to dryness under reduced pressure; the oily residue was redissolved in pure light petroleum. This extract, which contained the whole of the carotenoids of the skin, was clear and greenish-yellow in colour. Its absorption spectrum, measured with a Hilger visual spectrophotometer, showed maxima at 484 mμ and 455 mμ, and a minimum close to 472 mμ. When partitioned between petroleum ether and 90 per cent, methyl alcohol about half the pigment was hypophasic, indicating the presence of some free hydroxy-carotenoids. The hypophasic fraction was returned to petroleum ether and the total extract then adsorbed on to a column of magnesium oxide (B.D.H. preparation, low in arsenic) and developed with petroleum ether containing 2 per cent, of methyl alcohol. Two bands of pigment were obtained; a lower one, lemon-yellow in colour, with sharply defined boundaries (I) and a more strongly adsorbed diffuse one, which was pink (II). Bands I and II were eluted separately with excess solvent and returned to petroleum ether. Partitioned again with 90 per cent, methyl alcohol, band I proved to be the epiphasic and II the hypophasic fraction. Band I was then saponified for i hour with 6 per cent, alcoholic KOH at 60° C. and recovered in petroleum ether by diluting with an equal volume of water and extracting exhaustively. The saponification residue was then acidified with a few drops of glacial acetic acid and once more extracted with petroleum ether, but yielded no further pigment. The pigment recovered after saponification was found now to be hypophasic.

The absorption spectra of the two carotenoid fractions in pure hexane were almost identical, with maxima at 475mμ., 447 mμ, and about 425 mμ. These correspond closely with the absorption spectrum of lutein (fig. 1). Finally the two fractions were once more combined, adsorbed on to a column of magnesium oxide, and developed as before. Only a single band was obtained, which resembled band II of the previous experiment.

FIG. 1.

Absorption spectrum of total carotenoid from the skin of the ammocoete (continuous line) compared with that of pure lutein (dotted line). Solvent, hexane. (For ease of comparison the curves have been adjusted to the same value of optical density at the absorption maximum at 447 mμ.)

FIG. 1.

Absorption spectrum of total carotenoid from the skin of the ammocoete (continuous line) compared with that of pure lutein (dotted line). Solvent, hexane. (For ease of comparison the curves have been adjusted to the same value of optical density at the absorption maximum at 447 mμ.)

This analysis shows clearly that the skin of the ammocoete contains a single carotenoid, which is almost certainly lutein, about half of it being esterified and the rest in the form of the free hydroxy-carotenoid. Although lutein occurs in the eyes of many animals, there is no evidence that it plays any direct part in photosensitive processes, and it is also a common pigment of the skin of lower vertebrates in general. If, however, it is concerned in any way with the photoreceptors of the ammocoete, one would expect to find the greatest concentrations in those regions which are most sensitive to light. Portions of skin were taken therefore from the head, mid-body region, and tail, and the total carotenoids of each region estimated by the colorimetric method described by Steven (1948). The results presented in Table 1 show that there is no marked difference in the amount of lutein in the tail compared with other parts of the body.

TABLE 1.

Concentration of Carotenoid in the Skin, estimated as Lutein

Concentration of Carotenoid in the Skin, estimated as Lutein
Concentration of Carotenoid in the Skin, estimated as Lutein

The amounts of carotenoid extracted from the skinned bodies were negligible. Some extracts contained traces of yellow-green pigment, but not sufficient for further analysis. The liver, however, contained appreciable amounts of a hypophasic carotenoid, which appeared to be similar to the pigment of the skin, and in addition a small unsaponifiable fraction, which was probably carotene. The amounts obtained were insufficient for quantitative estimation or for further analysis.

The distribution of cells containing carotenoid was studied in preparations of fresh skin mounted flat and in frozen sections. Frozen transverse sections were cut from pieces of the tail and body which had been fixed in Baker’s (1944) formal calcium for about 12 hours in darkness, and subsequently washed for an hour in running water. Previous experience with other species of fish had shown that carotenoids in cells do not fade to any great extent in this time, provided the material is kept out of the light. Groups of rounded or ovalshaped cells containing greenish-yellow pigment were seen in most sections. They were most numerous in the sub-dermal tissue dorsal to the spinal cord in the base of the dorsal fin, and between the muscle blocks in the base of the ventral fin. In both these regions they were surrounded by and intimately associated with dense concentrations of melanophores. A few cells of the same type were scattered in the sub-dermal tissue surrounding the myotomes, but none were seen in the epidermis. They coloured intensely with Sudan IV and sudan black (fig. 2), and were decolorized almost instantly by acetone and methyl alcohol. These properties are typical of lipophores containing carotenoid, and since no other appreciable concentration of pigment was found, there can be little doubt that the bulk of the lutein extracted from the skin was contained in these cells.

FIG. 2.

Lipophores in the sub-dermal tissues of the base of the tail. Frozen transverse section, coloured with Sudan black.

FIG. 2.

Lipophores in the sub-dermal tissues of the base of the tail. Frozen transverse section, coloured with Sudan black.

The lipophores of the ammocoete differ from those of most fish in that they do not appear able to disperse and concentrate the pigment in response to varying conditions of external illumination. They do not therefore act as chromatophores as do the melanophore cells. It was thought at first that they might be the photoreceptors, but this is unlikely since, apart from the question of their innervation which is dealt with in a later section, most of the lipophores are closely screened from changes in external illumination by dense concentrations of melanin. Moreover they were rather less numerous in the tail, where photosensitivity is greatest, than in more anterior regions of the body.

A second type of pigmented cell was seen in some fresh untreated preparations of ammocoete tails mounted flat in diluted Ringer’s solution. They were situated in the middle layers of the epidermis and distributed in loose clusters containing usually about three to a dozen cells fairly close together (fig. 3). They appeared round or oval, about 10 μ in diameter, which is rather smaller than the majority of typical epidermis cells among which they lay. They were faint yellow, almost a pale straw colour, and the contrast between them and the surrounding tissues could be greatly increased by observing them in blue or blue-green light. The position of the absorption maximum of the pigment was estimated approximately by examining the cells through each of the Ilford Monochromatic Series of filters and adjusting the intensity of the incident light by means of a graduated neutral wedge to compensate for the relative energy transmission of the different filters. It was found that when proceeding from shorter to longer wave-lengths greater contrast was obtained, which reached a maximum in the blue-green and green (Ilford Filter Numbers 603 and 604), and thereafter diminished sharply. The cells could not be seen at all in light of wave-lengths longer than about 550 (Ilford Filter Number 605). The absorption maximum of the pigment appears to be situated in the region 490 to 520 mμ. The cells did not colour with sudan IV or Sudan black, but the pigment was immediately destroyed by acetone or methyl alcohol. The pigment usually faded in about half an hour under continued illumination.

FIG. 3.

Surface view of a portion of unstained epidermis of the tail, showing a cluster of pigmented cells. (The pigmented cells lie in the middle layer of the epidermis among the large, round, mucus-producing cells. A portion of the honeycomb appearance of the surface of the epidermis at a slightly higher focal level is also shown.)

FIG. 3.

Surface view of a portion of unstained epidermis of the tail, showing a cluster of pigmented cells. (The pigmented cells lie in the middle layer of the epidermis among the large, round, mucus-producing cells. A portion of the honeycomb appearance of the surface of the epidermis at a slightly higher focal level is also shown.)

The only other type of pigmented cell seen in these preparations was the erythrocyte. They were rather larger and more orange in colour. The walls of the capillaries in which they were contained could also be seen. Moreover they lay at a deeper level in the sub-dermal layers and there appeared to be no blood vessels in the epidermis.

The sensory cells and nerves of the tail were studied in methylene blue and silver preparations. Methylene blue staining was carried out without any important modifications by the method described by Whiting (1948). Small pieces of tissue were stained in a 0·1 per cent, solution of Gurr’s special preparation for intra-vitam staining, which was prepared by diluting a 1 per cent, stock solution with 0·7 per cent, saline. The use of a saline medium prevented swelling and damage of the cells around the cut surfaces, which were very noticeable if the stain was made up in distilled water. The physiological saline solution appropriate for fresh-water teleosts, given by Pantin (1948), was sometimes used, but appeared to possess no advantage over a solution of sodium chloride of approximately the correct osmotic pressure. Coloured solutions were found to be more satisfactory than rongalit-reduced ones, and after a few trials were used exclusively. The best definition of sensory cells and peripheral nerves was usually obtained after 1 to 2 hours in the stain. The stained preparations were made permanent when desired by Whiting’s procedure and were either mounted directly in Canada balsam or sectioned at 36 μ.

For silver impregnation Holmes’s (1947) ‘silver on the slide’ technique was used with sections cut at 10 or 15 μ. Embedding and sectioning was done in Steedman’s (1947) ester wax, which was found to be much less damaging to the tissues than paraffin wax. Of various fixatives Carnoy’s gave the best selective impregnation of axons, but had the disadvantage of causing distortion and gave a poor histological picture. This was true of all fixatives containing a high proportion of alcohol. Bouin’s fixative gave a much better histological picture at the cost of some loss of contrast, since all tissues tended to take the silver more readily. This could be partly compensated by increasing the proportion of pyridine in the impregnating mixture, as recommended by Holmes. Nonidez’s alcohol-chloral hydrate, recommended by Whiting, was also used and gave rather better definition than Bouin’s, although not such good general fixation.

Two types of sensory cells were stained with methylene blue, the neuromast organs and individual round or oval cells distributed in clusters in the epidermis. The former, which have been described by Young (1935) and others, are of the open-pit type. They occur at intervals along both sides of the body close to the base of the dorsal fin to within 2 mm. of the tip of the tail. The neuromast cells are packed closely together and are elongated in shape, projecting through to the outer surface of the epidermis. The other type of sensory cells, however, was found only in the middle layers of the epidermis, and they were not closely packed. In size, shape, and distribution they resembled closely the pigmented cells described in the previous section. They were most numerous in the outer part of the tail beyond the melanophores and cartilaginous fin supports. They were innervated by fibres which ran in the sub-dermal tissue, penetrated through to the epidermis and there terminated. Each cluster of sensory cells appeared to be innervated by a single fibre, whose terminal branches ended in definite boutons on the surface of the cells, and in addition there were many free endings (figs. 4 and 5). These fibres could be traced for considerable distances in whole preparations mounted flat. Most of them ran diagonally backward and outward from the base of the tail towards the edges of both the dorsal and ventral flukes (fig. 6); that is from about the point where the body narrows into the tail to the region where the clusters of sensory cells were most numerous. They could not be traced centrally in these preparations beyond the point where they emerged from the mass of melanophores surrounding the myotomes and spinal cord.

FIG. 4.

Surface view of nerve terminations in the epidermis and a cluster of sensory cells stained with methylene blue.

FIG. 4.

Surface view of nerve terminations in the epidermis and a cluster of sensory cells stained with methylene blue.

FIG. 5.

Two sensory cells and nerve terminations in ttansverse section. Methylene blue. 24 μ.

FIG. 5.

Two sensory cells and nerve terminations in ttansverse section. Methylene blue. 24 μ.

FIG. 6.

Methylene blue preparation of the whole tail of a small ammocoete, showing the course of some of the sensory fibres. (Many of the fibres shown terminate at sensory cells which are not visible at this magnification.)

FIG. 6.

Methylene blue preparation of the whole tail of a small ammocoete, showing the course of some of the sensory fibres. (Many of the fibres shown terminate at sensory cells which are not visible at this magnification.)

Silver impregnation was used to trace the course of the spinal nerves and the branches of the lateral line nerves. The histological picture obtained differed in certain respects from that with methylene blue. The sensory cells were not differentiated from the other cells of the epidermis, and though nerve-fibres were seen to penetrate the dermis and enter the epidermis, their actual terminations were not distinguished. The lateral line nerves lay close together between the myotomes just dorsal to the spinal cord. They were closely surrounded and in some sections almost obliterated by dense concentrations of melanin (fig. 7). Although they run very close to the lipophores containing carotenoid, no fibres were seen connecting with the lipophores. They were traced into the tail beyond the last pair of neuromast organs to about 1·5 mm. from the tip. At this point each nerve appeared to contain about 50 axons enclosed within a fine perineurium. In the terminal sections the perineurium disappeared and the axons became widely separated, so that it was difficult to decide where the lateral line nerves ceased to exist as definite entities. Some axons were traced around the dorsal edge of the myotomes and then ventrally for some distance in the sub-dermal tissue, while others extended out towards the periphery of the dorsal fin, where they peneuated the dermis and entered the epidermis (figs. 8 and 9). All these axons ran in a general posterior direction from the lateral line nerves towards the periphery. Fig. 1o shows in diagrammatic form the general anatomical relations of the lateral line nerves and their branches in the tail.

FIG. 7.

Transverse section of the lateral line nerves in the base of the tail. Silver. 15 μ.

FIG. 7.

Transverse section of the lateral line nerves in the base of the tail. Silver. 15 μ.

FIG. 8.

Fibres of the lateral line nerve in the sub-dermal tissues of the tail. One axon is seen at the point where it penetrates the dermis (A). Silver. 15 μ.

FIG. 8.

Fibres of the lateral line nerve in the sub-dermal tissues of the tail. One axon is seen at the point where it penetrates the dermis (A). Silver. 15 μ.

FIG. 9.

Part of the course of a fibre of the lateral line nerve, showing the point where it penetrates the dermis. Silver. 15 μ.

FIG. 9.

Part of the course of a fibre of the lateral line nerve, showing the point where it penetrates the dermis. Silver. 15 μ.

FIG. 10.

Diagram to show the general anatomical relations of the lateral line nerves and their branches in the tail.

FIG. 10.

Diagram to show the general anatomical relations of the lateral line nerves and their branches in the tail.

Branches of the dorsal roots of the spinal nerves also run dorsally just inside the myotomes, passing very close to the lateral line nerves. It was not always easy to distinguish them from branches originating from the latter, though in general the spinal nerves contained larger numbers of fibres. It was not possible to trace branches from the lateral line nerves into the ventral lobe of the tail. No fibres were seen to run from them in a ventral direction inside the myotomes, and it seems likely therefore that they pass through the sub-dermal layer immediately under the skin.

The well-established physiological characteristics of the light reaction of the ammocoete provided a number of criteria in the search for a sensory system, and also imposed certain requirements. Although the whole skin of the animal is more or less sensitive to light, the tail is the most sensitive region, and it was to this organ that attention was mainly directed. Since the light sense is diffuse and the minimum intensity of illumination required to elicit a swimming response varies inversely with the area of skin stimulated, one would expect the receptors to be widely distributed but most numerous in the most sensitive regions. Furthermore, since there is a more or less continuous layer of melanin in the sub-dermal tissues, surrounding the myotomes, spinal cord, notochord, and viscera, visible radiation must be almost completely absorbed before it reaches these organs. It seemed likely therefore that the photoreceptors are situated outside the melanin layer in the epidermis or in the fins, where melanophores are few or absent altogether. Finally it was clear from Young’s (1935) experiments, which I have confirmed, that the light receptors are innervated by the lateral line system and not by spinal nerves.

The distribution of the sensory cells of the epidermis, which stained with methylene blue, agrees well with these theoretical requirements. Although some were seen in the body region, they were far more numerous in the tail, particularly towards the outer edge and well away from the melanin layer. There is little doubt that they are identical with the pigmented cells which were seen in fresh unstained preparations and with which they agree closely in size, shape, frequency, and distribution. Since the latter did not stain with sudan IV or sudan black, they cannot be regarded as lipophores in the usual sense although the colour was destroyed by lipoid solvents. It seems more probable that the pigment, if it was carotenoid in nature, was bound to a protein. The conditions under which it was observed make it unlikely to be itself the photosensitive substance responsible for the light reaction, but it could be a product of the primary photoreaction. Coloured derivatives of photosensitive pigments, such as the retinenes which are the primary product of the bleaching of visual purple, are often relatively light-stable and are themselves bleached only by higher intensities or by more prolonged illumination. The fact that the yellow colour of the epidermal cells faded during the course of prolonged observation under a strong light is consistent with this idea. The fading may, however, have been due to other factors, such as postmortem changes in the tissues, so that this line of inquiry cannot be pressed further at present.

Clearly if the sensory cells in the epidermis which stained with methylene blue are the photoreceptors concerned with the light reaction, the nervefibres which terminate on them should be connected with the lateral line nerves. This has proved to be most difficult to demonstrate, partly because of the differences in the histological pictures obtained with silver and with methylene blue and partly because branches of the dorsal root nerves of the spinal cord pass so close to the lateral line nerves that it is by no means easy to decide the origin of many of the fibres. Also degeneration experiments, in which the lateral line nerves were severed at a point just posterior to the last gill pouch and the ammocoetes killed a week or so later, gave inconclusive results. The operation could be performed only on very large ammocoetes, on which the silver impregnation method did not prove satisfactory. However, the following points have been established with certainty:

  1. The lateral line nerves extend to within 2 mm. from the tip of the tail and beyond the level of the last pair of neuromast organs.

  2. Each lateral line nerve contains about 50 or more axons at a point beyond the last pair of neuromast organs.

  3. In addition to the branches to the neuromast organs, other branches of the lateral line nerves run in the sub-dermal tissues towards the outer edge of the tail and penetrate through the dermis into the epidermis.

  4. Although no sensory, cells were identified in silver preparations, the lateral line branches traced into the tail were similar in number and distribution to those seen in methylene blue preparations.

  5. Most of the methylene blue stained fibres connecting with the sensory cells radiate from about the base of the tail, from about the point where the lateral line nerves are dispersed into a number of small branches.

No evidence was obtained that the pigmented cells in the sub-dermal tissues which contain carotenoid are concerned with the photoreceptor system, nor do they appear to play any part in the colour changes of the animal. The only colour change in L. planeri appears to be a relative darkening and lightening of the skin brought about by expansion and contraction of the melanophores. The lipophores clearly take no part in this, and it is difficult to assign any function to them other than the storage of fats and carotenoid. This is certainly not the case, however, in all lampreys. According to Wild (1903),L. marinus exhibits a great range of colour variation, changing from yellow to a marbled pattern of blue and white patches, which suggests that the species possesses expansible xanthophores or similar types of chromatophore in which carotenoids are the most probable type of pigment. It is possible that the condition found in L. planeri is a neotenous feature, in which case the pigmented lipophores may be regarded as a larval type of xanthophore. The development of chromatophores in other species of fish provides some evidence in support of this point of view. In the trout, for instance, the xanthophores and erythrophores pass through a stage in which they resemble closely the lipophores of the ammocoete, while the melanophores, which develop precociously, are already fully functional (Steven, 1949).

In addition to the principal subject of inquiry this work has provided some new information on the sensory innervation of the lamprey’s skin, an aspect which has received rather little attention from the numerous workers on the nervous system of this animal. Free nerve endings in the epidermis of L. planeri have been described by Stefanelli (1932) and by Boeke (1934), and can be demonstrated either with silver or with methylene blue. According to Stefanelli, who used methylene blue, the fibres ran in the melanophore layer of the sub-dermal tissues, turned sharply to pass perpendicularly through the dermis, and divided into many terminal branches in the epidermis. Each branch ended in a definite bouton in the middle layers of the epidermis, giving a characteristic ‘candelabra’ appearance to the whole structure, and each fibre served a clearly defined area of the skin, which did not overlap the area of adjacent fibres. No sensory cells were distinguished, and the central connexions of the fibres were not traced. Tretjakoff (1927) described terminations of the VIIth cranial nerve on the neuromast cells of lateral line organs in the head region, which appear very similar to the terminations I have found on the sensory cells of the tail. The differences between them appear to be of degree only; the neuromast cells being more closely packed, the terminations are rather more concentrated and there are fewer free endings.

In conclusion it is relevant to compare the sensory system of the ammocoete with corresponding structures of invertebrates which exhibit similar types of light reaction. Unfortunately the amount of information available on the behaviour of lower organisms in response to light greatly exceeds critical studies on the structures concerned with those reactions. It is clear, however, that many of the pigmented spots and various organs described as ‘eyes’ or ocelli found in annelids, molluscs, and tunicates are not photoreceptors at all. Hecht (1918), for instance, showed that the light-sensitive region of Ascidia atra is a vaguely defined area about 1·0 to 1·5 cm. from the tip of the siphons, while the yellow spots on the rim of the siphons are quite insensitive. Nor is there any evidence that the ocelli of dona intestinalis described by v. Haffner (1933) are sensitive to light. The sensory cells of these structures possess hairs, which are characteristic of receptors that respond to mechanical deformation and the whole structure appears similar to the simpler types of lateral line organs of vertebrates. The structures in the siphons of Mya arenaria and in the skin of Lumbricus terrestris, described by Light (1930) and Hess (1925) respectively, are more convincing, and resemble in many respects the sensory system of the ammocoete. According to Light the photoreceptors of Mya are oval cells about 9 to 15 μ in diameter, scattered in loose clusters in the epithelium lining the siphons. They possess axons which join the main longitudinal nerves connecting with the visceral ganglia. In Lumbricus and other earthworms similar cells are found in the epidermis, particularly in the prostomium and the dorsal side of the anterior segments of the body, and are connected by numerous nerves with the suprapharyngeal ganglia. Section of some of the nerves or removal of the ganglia modified but did not abolish the light reaction. As in the ammocoete the sensory cells of both Mya and Lumbricus are most numerous in those parts of the animal which are most sensitive to light. They are also arranged in loose clusters and are widely distributed in the skin. In both invertebrates, however, the axons are a part of the sensory cells, whereas the arrangement in the ammocoete is typical of a vertebrate; the cell bodies of the axons are situated in the central nervous system and there are definite bouton terminations at the sensory cells. Another difference is that the cells of Mya and Lumbricus contain a prominent refractile body, termed an ‘optic organelle’, which is not apparent in the ammocoete. In my methylene blue preparations the nucleus was visible as a relatively clear area, but no other intra-cellular structure was distinguished.

I wish to thank Professor James Ritchie for his interest and encouragement and for his kindness in reading this manuscript.

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