Further Microscopical Details of New Zealand Loricata.
[Read before the Auckland Institute, 30th July, 1929; received by the Editor, 15th July, 1929; issued separately, March 31st, 1930].
Although the primary object of this paper is to record certain observations on the histology of the nerve endings present in the tegmentum of Loricate shells, the physiological aspect of the subject is so intimately associated with it that it is impossible to study these minute organs without speculating upon their function, and striving to formulate a working hypothesis accounting for their existence and utility.
There are three distinct organs, ocelli, megalaesthetes and micraesthetes; though these were recognised and described more than forty years ago, their correlative values have never been thoroughly understood. The most highly evolved of these nerve terminals is the ocellus, of which Moseley has written an excellent and comprehensive description in his paper, “On the Presence of Eyes in the Shells of Certain Chitonidae, etc.” Quart. Journ. Micr. Soc., Lond., vol. 25, 1885. Of the New Zealand chitons, only four genera are found to be ocelliferous, namely, Paricoplax, Icoplax and Eudoxochiton of the family Lepidochitonidae, and Onithochiton of the family Chitonidae. Other genera have been credited with possessing eyes, but as they have been definitely proved to be alien to our marine fauna, there is no need to specify them.
The development of the ocellus may be studied with better results in Eudoxochiton than is the case in other allied genera, for the eye is met with in several distinct stages of growth. The first of these is a slight advance upon the most rudimentary form of a visual organ, which is represented by a mass of photoscopic pigment surrounding a nerve terminal. A superficial resemblance to this form is seen in the decalcified shell of young specimens of Eudoxochiton where it appears as a series of rather irregularly shaped clumps of blackish-brown pigment, arranged in quincuncial formation upon the lateral areas of the median valves, upon the whole of the anterior valve, and on the post-mucronal area of the posterior valve. Each mass occupies the centre of a lozenge-shaped loculus formed by a geometrical segmentation of the stroma (Fig. 1). For the most part the pigment is closely aggregated in a compact amorphous mass, rather deeply embedded in the stroma which is transparent and, as a rule, structureless. In a few instances a small cloud of minute granules can be seen detached from the main body, a preliminary step in the dispersion of the pigment which eventually spreads in the containing wall of the eye capsule. In unison with this dispersion of pigment, the bulb of the eye becomes enlarged, pushing aside the adjacent lines of micropores and, gradually emerging through the stroma, forms a clear space in the centre which becomes the lens. In one prepared specimen,
where the pigment clumps have slipped aside owing to the pressure of the cover glass, nearly the whole outline of the eye capsule is plainly visible. The lens now shows a definite circular outline, though not as yet free from pigment (Fig. 2). To examine the final stage the whole shell must be used, not decalcified as those with which we have been dealing. It must be ground on the under-surface to ensure the requisite degree of translucency and preferably stained with some simple aniline reagent. It will now be seen that quite half the total
number of eyes are still immature. They are covered with a pale yellow periostracum and lack the highly refractive property of the perfect organ. As the ocellus attains maturity it becomes more convex, causing thinning and atrophy of the periostracum. Finally, that membrane disappears, leaving the cornea free to admit the maximum amount of light. When this stage is reached the ocelli are highly refractive and, by transmitted light, sparkle with a wonderful brilliancy (Fig. 3). There is also a marked difference in the appearance of the microporal tubules. Those connected with the mature ocelli are very conspicuous as they traverse the ridges forming the fine sculpture of the valves, while those in the vicinity of the immature ocelli are barely visible. An explanation of this condition will be found in connection with the ocelli of Paricoplax.
The ocelli of Paricoplax are very small, and at a casual glance are scarcely recognisable as eyes. However, they have precisely the same quincuncial arrangement and distribution as those of Eudoxo-
chiton, radiating from the apex of the anterior valve, disposed in a phalanx on the lateral areas of the median valves, with the point at the mucro, and widening out towards the lateral border, and also radiating from the mucro to the margin of the post-mucronal area of the posterior valve. They appear under a hand lens merely as minute black specks, but when examined under a power of about 150 diameters some of them are seen to have a well developed cornea in the middle, somewhat retractile and showing a pale reddish tinge such as occurs in Onithochiton, a genus possessing the largest and most advanced type of eye of any New Zealand chiton. The reddish tinge in Paricoplax will be found on further examination to be due to refraction in the superimposed stroma, for it is quite absent in the intraocular media as viewed through the transilluminated cornea in specially prepared specimens. This will be referred to later, as at present we are dealing with the natural shell. The black pigment is disposed in a ragged and untidy fashion round the eye, and in the majority of cases obscures the cornea in varying degrees. The transparent stroma is noticeably thicker in this genus, and the pigment is seen both on the surface of the tegmentum and through the depth of the stroma down to the level of the corneal margin. This is a totally different condition from that which obtains in Eudoxochiton, where the eyes project beyond the general surface of the tegmentum, and the pigment in the adult eye is limited to a very narrow black line clearly defining the eye capsule. It is well known that in the living animal the valves of Paricoplax and juvenile Eudoxochiton are coated with a tenacious glutinous epidermis, and this must be removed before a satisfactory view of the mature ocelli can be gained. The valves to be examined must therefore be first boiled in a 5% solution of caustic soda for ten minutes, followed by several changes of fresh water, and afterwards stained in an aqueous solution of gentian violet. The inspissated epidermis is almost entirely removed except for a few isolated patches, which are readily recognised by their deeper staining and a curling up of the edges. In the clear portions the ocelli show up well. They are smaller than those of Eudoxochiton, though very similar in appearance, and the scheme of development is precisely the same. Upwards of 8,000 ocelli have been counted on one small specimen no larger than twelve millimetres in length. Full grown specimens have between twenty and thirty thousand of these organs. In decalcified specimens of Paricoplax the development of the eye can be traced from the pigment-charged megalaesthete to its final stage, and it will be noted that every single megalaesthete in the ocelliferous areas ultimately becomes an eye. At first the megalaesthete is seen to be of the usual fusiform shape with a deposit of pigment situated in the upper quarter (Fig. 4). Then the body of the megalaesthete commences to swell, and at the same time the pigment expands. The swelling continues, particularly at the shoulder and under-surface of the body of the organ, until the megalopore is seen to be below the level of the shoulder. The micraesthetes, by the extension upwards of the wall of the megalaesthete, are lifted higher and show a tendency to group themselves about the megalopore. The expansion of the pigment, the gradual appearance of the lens, and the actual shaping of the eye capsule, being the same as in Eudoxochiton, require no
further description. One interesting incident cropped up in the preparation of a decalcified specimen which deserves mention. The pigment granules were observed to have worked their way along the
ducts of the micraesthetes (Fig. 5). It is possible that this migration may have been accidental, and due to the pressure of the cover glass; but, whatever the cause, it proves conclusively that the megalaesthete is a hollow organ, that the pedicels of the micraesthetes are permeable tubes, and that the pigment is primarily produced within the megalaesthete. On the other hand, this disposition of proliferating pigment may be a normal process, thus accounting for the darker micraesthete tubules of Eudoxochiton previously referred to.
Fig. 5.—Paricoplax platessa Gould. Lateral area (decalcified). Pigment granules in micraesthete tubules.
The stroma which has been alluded to several times demands attention. It is a homogeneous colloidal substance seen in decalcified shell. It serves as a cement tissue in which lie embedded all the canals, nerves, and nerve terminals of the tegmeutum. In the living shell it is impregnated with lime salts. By some writers it is termed the cuticula, by others the stroma, while Moseley made use of both
terms indiscriminately. In my opinion a distinction should be made, for in decalcified shells of Eudoxochiton and Paricoplax a thin delicate membrane can be separated from the surface of the tegmentum, which on microscopical examination is seen to include terminals of the megalopores and micropores, spaced out in the normal regular pattern. For this membrane the term cuticula is quite appropriate, and the term stroma should be reserved for the connective interstitial tissue lying beneath it. At the same time it must be pointed out that this is an artificial rather than a structural distinction. The cuticula may be aptly compared to the skin which forms on the surface of boiled starch when it cools.
In the genus Onithochiton the eyes are remarkably well developed. They are large enough to be discerned by the naked eye, standing out as shining black dots, situated in all the usual regions wherein these organs are met with (Fig. 6). Vertical sections of these eyes have been made showing their internal structure, but much may be learned from an external examination. The fully developed eye is about 0.04 mm.
across the cornea, round in outline, and by transmitted light a bright ruby colour, sharply defined by a border of dense black quickly fading away in the depth of the stroma. This surrounding pigment is present for the purpose of preventing irradiation, and is quite different from that of the eye capsule, both in colour and texture. The cornea is a concavo-convex transparent structure, through which one can distinguish, by careful focussing, a small circle centrally framed in a slightly darker border. This is the lens in the pupillary aperture of the eye capsule. It is preferable that a low power such as a ⅔ inch objective be used, otherwise blurring of the image is caused by refraction. The whole of the eye capsule can only be examined in the decalcified shell, when, if the process be not carried too far, the cornea remains practically unaltered. The capsule is a transparent golden
brown pyriform or bulbous hollow body tapering away to a stalk or canal for the transmission mainly of the optic nerve which reaches its destination from the incisura in the margin. It will be noted that the number of ocellar rays is in direct proportion to the number of valve slits in all ocelliferous species. The capsule itself is not affected by dilute mineral acids and is generally considered to be of a chitinous nature. Towards the external margin the eye capsules are not so advanced as are those nearer to the apex. They are sickle-shaped or crescentic with the unfinished ends directed outwards, or globular and lacking the stalk. Later on they become truly pear-shaped with the canal gradually increasing in length. The lens makes its appearance before the capsule is fully formed. It must not be assumed that the ocelli originate only at the external margin of the valve. Specimens of Onithochiton which have been decalcified and properly stained clearly show at some distance from the external margin ocelli in the earliest stages, that is before any trace of pigment in the eye bulb appears. These aborted forms are of paramount interest, for they
Fig. 7.—Onithochiton negleotus Rochebrune. Lateral area (decalcified) megalaesthetes changing to ocelli.
reveal, step by step, the whole process of development in the most minute detail. The first change to be noticed is a narrowing of the proximal end of the megalaesthete, the distal portion broadens out, rising and extending beyond the neck, with the micraesthetes crowding to the upper part (Fig. 7). These next arrange themselves horseshoe fashion round the megalopore which presently disappears. The capsule then assumes the typical pegtop form and the pigment is observed for the first time as a sickle-shaped arc at the upper pole. This arc increases inwards, the horns creep round until they finally meet, and lastly the neural canal extends until it equals in length the
bulb itself. Pari passu with these developments, the micraesthetes with shortened canals have settled round the periphery and on the surface of the bulb in much the same manner as the nerve terminals are distributed round the sheath of a hair follicle.
The most striking feature of Onithochiton is the enormous number of permanent megalaesthetes, covering as they do the entire field of the ocelliferous areas (Fig. 8). This is in marked contrast to the peripheral nerve scheme in the Lepidochitonids where in corresponding areas there is not a single megalaesthete to be found in its original state. Each one has been converted more or less completely into an ocellus. It is needless to say that the megalaesthetes in all central areas are invariably unchanged.
That the function of the megalaesthetes, over and above the part they play in the development of ocelli, is to secrete a mucoid or glutinous material, or perhaps merely to excrete moisture, may be concluded from the following facts: (a) the large size of the megalaesthetes in the two genera, Eudoxochiton and Paricoplax, which, when living, have their valves conspicuously coated with a glutinous cuticle; (b) the flask-like shape of the organ, which resembles in appearance an efficient and highly elaborated goblet cell; (c) the fact that the contents of the organ in decalcified specimens fill the interior in many instances, while in others the megalaesthete is almost completely empty. The granular nature of the contents is strongly suggestive of mucinogen; (d) the action of a reagent entering the mouth of the megalopore and staining the contents, observed both in the whole and the decalcified shell. This fact is strikingly displayed in Sypharochiton sinclairi in a decalcified portion of the central area of a valve where the apices of the papilliform bodies stain very deeply with methylene blue, while the lower parts stain less freely (Fig. 9).
At this point it is interesting to note that Moseley, although he was unable actually to demonstrate the fact, was of opinion that the
megalaesthetes are capable of protrusion and retraction, a condition compatible respectively with dilatation and contraction of the body of the organ; dilatation being commensurate with the accumulation of secretion within the glandule and contraction coincident with the evac-
Fig. 9.—Sypharochiton sinclairi Gray. Central area decalcified). Papilliform bodies in megalaesthetes.
Fig. 10.—Acanthopleura spiniger (decalcified). Striated megalaesthete (from Moseley's figure).
uation of the contents. “Some of the component strands of the body of the megalaesthetes” he states “show a transverse striation, while others are not striated. They bear nuclei at intervals.” He gives a figure, here reproduced (Fig. 10), of the megalaesthete of Acanthopleura spiniger, but makes no comment bearing upon their significance. I have observed the same in Eudoxochiton (Fig. 11) and regard them
as fibres of a contractile character, having the same function as ordinary muscle tissue. Onithochiton also shows this transverse striation of the megalaesthete, and no doubt a similar condition will be found in the majority of chitons. So far I have only been able to verify this fact in vertical sections of the valves, where one half of the body of a megalaesthete has been sliced away. The striations can then be observed in the interior of the remaining half. They are extremely small in Chitonidae, and would have escaped notice had it not been for some larger corrugations running round the lower and outer surface of the goblet. Between the goblets and springing up from below are numbers of dome-shaped bodies of various sizes. These are the young megalaesthetes, each separately enclosed in a delicate tunic, with the attendant micraesthetes packed neatly together so as to occupy the least possible space (Fig. 12).
Fig. 12.—Onithochiton neglectus Rochebrune. Lateral area (decalcified). Striated megalaesthetes, partly in section, and brephic buds of same.
The protusile faculty of the megalaesthete is the natural sequence of the unyielding nature of the calcareous matrix of the tegmentum in which it lies, for when the megalaesthete is fully distended expansion can only take place by a bulging of the free extremity through the bottle-neck opening on the surface of the shell. On the discharge of the contents the glandular body resumes its normal tension, and the trumpet-shaped neck is retracted to the level of the tegmentum. The rigidity of the calcareous matrix further necessitates the provision of some form of protection for the end of the megalaesthete, hence the infundibular orifice, which when withdrawn into the tapering neck of the megaloporal pit becomes automatically closed. In decalcified specimens of Amaurochiton glaucus the interior of the mouth of the megalaesthete can be seen distinctly funnel-shaped (Fig. 13a). The outer rim comes into view first; then by careful focussing, and manipulation of the substage condenser, the concentric rings appear one after the other, until finally, at the base of the funnel, is seen a minute circular disc which is the apex of the papilliform body. Whether this little disc is perforated or not it is impossible to say, nor is it essential, but that it is pervious is highly probable, allowing a process analogous to diapedesis in blood capillaries to take place. The best results are obtained by employing a 2 per cent, solution of nitrate of silver which produces a rich dark brown stain with good contrast and
clear detail. Within the megalaesthete is a delicate homogeneous lining separable at the upper portion and presenting the appearance either of an acuminate sac or of a tapering truncated tubule extending upwards (Fig. 13b and c).
When in the acuminate sac-like form the apex barely reaches the neck of the megalaesthete, but the tubular form extends well within the neck. In the former condition the granular contents fill the whole of the sac, giving origin to the name papilliform body. The tubular form is assumed when the sac is comparatively empty. Another point to be taken into consideration is the fact that the circular rims of the megaloporal and microporal apertures are actually incorporated with the cuticula as we have already observed in the decalcified shell. In the calcareous shell we thus find fixed points of insertion to assist in the filling and emptying processes of the megalaesthete. This structural feature also explains the glittering bead-like appearance of the megalopores when a living chiton is examined under the microscope. That an exudation or sweating does
Fig. 13.—Amaurochiton glaucus Gray. Central area (decalcified). a Infundibular megalopore. b Acuminate papilliform body, c Tubular phase of same.
take place from the valves is certain. I have kept Sypharochiton pellisserpentis, notoriously emergent forms, in a jar of sea-water. They promptly crawled up the sides of the glass and stayed on the under surface of the lid. After the water was removed, the animals lived for more than a week and the shells remained moist during the whole of that time. Dr. W. Marshall (l.c.) regarded the entire canal system as being tubular and respiratory in function, and I am inclined to believe he was correct to a certain extent. Moseley opposed that theory and affirmed that the megalaesthetes were homologous with the eyes. This we have proved to be true so far as a select number of these organs is concerned, but we have still to reckon with the vast number of permanent megalaesthetes, which do not, and were never destined to, become eyes.
Though the foregoing facts furnish fairly conclusive evidence that the extra visual function of the megalaesthetes is more than that of mere tactile corpuscles, as formely supposed, the question still hovers in the domain of philosophic conjecture. On the other hand, a tactile or rather thermoscopic function may be safely assigned to the micraesthetes, not only on account of their enormous number—they outnumber all other nerve terminals by fifteen to one on an average—but by reason of their simple structure and universal distribution.