The Green Bodies of the Intestinal Wall of certain Chaetopteridae. By C. Berkeley.

With Plate 26.

INTRODUCTION. CLAPAREDB (1) seems to have been the first to attribute the colour of the intestinal wall of Chaetopterus variopeda- t u s Eenier to the presence of small spherical green granules in the epithelial cells of the tissue. The distribution of the granules in the cells was subsequently described by Laffuie (2) and by Lankester (3). These three authors considered that they con- sisted of a pigmented cell secretion concerned in one way or another with digestion. Brandes (4), on the other hand, expressed the view that the granules were not to be looked upon as a metabolic product of the worm, but were organisms of an algal nature analogous to those which had previously been described living symbiotically or parasitically in various animals by Brandt (5) and others. He based this view on the general appearance of the granules as figured by Lankester and on the similarity between the absorption spectrum of the green pigment chaetopterin extracted from them and that of chlorophyll. Enders (6), commenting on this difference of view, suggests that, if Brandes is correct, it should be possible to cultivate the green organism outside the worm as I'amintzin (7) and Beijerinck (8) did in the case of Zoochlorella from Hydra viridis. Since that time many instances of algal and animal associa- tions have been recorded, but no conclusive evidence appears to have been brought forward in the case of Chaetopterus. 466 C. BERKELEY The common occurrence of three species of Chaetopteridae, each representing a different genus of the family, in the im- mediate vicinity of the Biological Station, Nanaimo, B.C., in each of which the intestinal tract is visible through the body- wall of the abdominal region as a well-defined deep green tract, suggested that some information on the nature of the pigmented bodies in the epithelial cells might be obtained by a comparative study. The three species are : (1) Mesochaetopterus taylori Potts; (2) Phyllochaetopter us prolifioa Potts ; and (3) Leptochaetopterus pottsi Berkeley. They have all been recorded, and the first two described, by Potts (9) ; the third, only briefly mentioned by him and attributed to the genus Telepsavus, was described more fully by my wife (10). Mesochaetopterus and Leptochaetopterus inhabit tubes which run a considerable distance into the sand-banks in which they are found, the former frequently two feet or more in an almost vertical direction and then as far in a more horizontal one, the latter about half these distances. In neither case does the posterior end of the tube emerge to the surface as in that of Chaetopterus variopedatus. The animals are usually at least half the length of their tubes, and the abdominal region makes up by far the greatest part of their length. Phyllochae- topterus is found in tubes occurring in matted masses on gravelly bottoms at a depth of 15 to 20 fathoms, and is obtained easily in the dredge. Potts points out that it has the interesting faculty of increasing asexually, posterior portions of individuals splitting off and regenerating the anterior regions. He did not observe the sexual forms, but these are occasionally found. Preliminary examination showed that the green coloration of the intestinal wall was due in all three cases to spherical granules very similar to those described by Lankester from Chaetopterus variopedatus. A pigment having the pro- perties of chaetopterin could be extracted from each of them. It is easily soluble in strong hydrochloric acid (50 per cent. ' pure ' HC1), in which it seems to be quite stable even on prolonged boiling, differing thus very essentially from chlorophyll. No GREEN BODIES OF CHAETOPTERIDAE 467 phaecocyanin, or other water-soluble pigment, could be extracted from the green tissue. The tissue from each of the three species was examined by means of sections and the green bodies from teased-out material by hanging drops and dry films.

SECTIONS. The green colour of the intestinal wall remains permanent in material fixed in sublimate and bichromate (Zenker's mixture) or picro-formol (Bouin). In transverse sections of the abdominal region of Mesochaetopterus or Leptochaetopterus thus fixed the intestinal wall is seen at low magnification to con- sist of a heavily pigmented, much convoluted, tissue. The intes- tine occupies a larger part of the abdominal cavity than in Chaetopterus variopedatus, and the lumen is much smaller in relation to the thickness of the wall (fig. 1, PI. 26). Much the same condition exists in Phyllochaetopterus, but in this case the intestinal wall is relatively thinner and less convoluted and the lumen larger. At higher magnification the intestinal wall is seen to be built up of the usual two layers of cells, the columnar tissue being similar to that figured by Lankester and others for Chaeto- pterus variopedatus. In sections taken anywhere except near the anal end the cells of this tissue are crowded with spheri- cal green bodies varying in diameter from 1 to 8/x (fig. 2, PL 26). In Mesochaetopterus they are usually an olive shade, but some variation occurs in the depth of colour. In Leptochae- topterus it is commonly lighter and more blue-green. In both these species the green bodies are as a rule quite homogeneous and no structure can be brought out by staining. Quite rarely a few individuals have been seen in the sections showing a granular structure. A circular area rather more refractive than its sur- roundings, seen in some of the smaller and more lightly coloured green bodies, was at first taken for a nucleus, but it could not be differently stained. I shall refer to this later. In Phyllochae- topterus there is more variation in the colour of the green bodies than in either of the other species, some of them approxi- 468 C. BERKELEY mating to a blue colour, and a considerable number of them show distinct granular structure (fig. 3, PI. 26). The granules are accentuated in sections stained with both nuclear and cyto- plasmic stains. In addition to the more or less uniformly dis- tributed granules a roughly circular area staining selectively with haematoxylin occurs in some of the green bodies and may be a nucleus. In sections taken near the anal end of the annelids a different condition is found. Here the green bodies are uniformly smaller than in the more anterior region of the abdomen, averaging no more than 2/x in diameter and are not distributed throughout the epithelial cells. The majority are concentrated at the ends which border on the lumen, but a few are found scattered through the cells, usually lying immediately adjacent to the cell-wall (fig. 4, PL 26). The nearer the anal end the section is taken the fewer are the green bodies in the cells, until, finally, they are entirely absent from the last few segments. In the course of his studies of the development of C h a e t o - pterus variopedatus, Enders found that the green colour of the digestive tract could be observed in the larvae at an early stage, and that by the time they had attained a length of 2 mm. it had become quite marked, though less so than in fully trans- formed larvae. Free-swimming chaetopterid larvae of about this length have been taken over the beds in which Mesochaeto- pterus and Leptochaetopterus occur, and probably belong to one or other of these species. In these larvae the colour of the digestive tract was quite apparent and sections showed that it was due to green bodies in the epithelial cells precisely similar to those found in the adults. The conditions of size and of density of distribution in the cells resembled those found in sections taken near the posterior end of the adult animals. In material fixed in acid alcoholic fixatives the pigment is entirely removed from the green bodies. A fixative consisting of 95 per cent, methyl alcohol (100 parts), water (20 parts), formalin (5 parts), and glacial acetic acid (5 parts) was used for this purpose. Pieces of abdomen of Mesochaetopterus left in this fixative until decolorization was complete (some GREEN BODIES OF CHAETOPTERIDAE 469 months) were stained in bulk in borax-carmine. Sections showed stained, but shrunken and deformed, residues of the formerly green bodies in the epithelial cells. More satisfactory results were obtained by fixing for a short period in the alcoholic fixative and completing the decolorization, in the sections, with alcohol containing hydrochloric acid (10 drops cone. HC1 to 100 cc. 95 per cent, alcohol). In sections so treated the green bodies were much less deformed. They could be stained with any of the ordinary cytoplasmic stains. Lankester mentions a ' colourless stroma ' which remains after the green bodies in Chaetopterus variopedatus have had their pigment removed, and this corresponds without doubt with the de- colorized residuum in my sections. The fact that such a stain- able residuum remained after decolorization seemed to indicate that the green bodies had something of the nature of a bounding membrane or cell-wall, and to be opposed to the view that they consisted of a cell secretion. Sections of material fixed in osmic acid fixatives showed that the green bodies contain a quantity of oil. They are stained intensely black, whilst the remainder of the contents of the epithelial cells is uncoloured.

HANGING DROPS. The green bodies can be easily separated from the tissue containing them by teasing it out in water, filtering through fine bolting silk, and fractionating the filtrate by means of a centrifuge. A great deal of oil is present in this filtrate, but it remains in suspension on centrifuging, and after a few washings a residue is obtained consisting of little but the green bodies and bacteria. Examination of such material, freshly isolated from P h y 11 o - chaetopterus, in a hanging drop showed immediately that the green bodies were discrete organisms. A large number of them were seen to be in active independent movement, and fiagella could usually be detected on the motile individuals. A trace of basic fuchsin added to the drop brought these out quite clearly. Two flagella occur on each individual, and they are of 470 C. BERKELEY approximately equal length, at least twice as long as the dia- meter of the organism bearing them (fig. 5, PI. 26). They are carried on a minute protuberance which appears as a bright spot when the organism is swimming in such a position that it is seen through the green body. This is without doubt the refractive • area previously referred to as having been seen in sections of Mesochaetopterus and Leptochaetopterus, and taken for a nucleus. Both homogeneous and granular individuals, of all sizes except the largest, showed motility. The organisms in a fresh suspension prepared from the anterior part of the intestine of Mesochaetopterus or Leptochae- topterus showed no motility, but after standing in a cool room for twenty-four hours, many motile individuals were present and flagella could be detected. After forty hours the majority of the green cells were in active movement. In the case of Leptochaetopterus some granular cells, similar to those seen in Phyllochaetopterus, were present, and these showed motility as freely as the homogeneous forms. In the material from both Mesochaetopterus and Leptochae- topterus almost colourless cells of the same size and shape as the green bodies occurred and were in many cases motile and flagellated. The flagella are the same length, relative to the individual organism bearing them, as in Phyllochaeto- pterus, and are similarly carried. The organisms from the three species of annelid cannot in fact be differentiated except to some extent by colour and by the greater frequency of granular forms in the case of Phyllochaetopterus. If the green bodies are isolated from segments near the anal end of Mesochaetopterus or'Phyllochaetopterus, actively motile individuals are found amongst them immediately. They are of smaller size in this case, averaging about 2 JU. in diameter, and no structure can be made out. A tendency to greater frequency of motility, when freshly extracted, amongst the green bodies taken from near the tail than amongst those taken further anteriorly was subsequently found to exist in Phyllochaetopterus also, particularly in the longer sexual individuals which are found occasionally. In the usual asexual GEEEN BODIES OF CHAETOPTERIDAE 471 forms the abdominal region is "short, and since they are constantly being budded off the anal end of the parent stock, the anterior abdominal segments are of recent growth compared with the corresponding segments inMesochaetopterus and L e p t o- chaetopterus. This greater approximation in age of anterior and posterior segments in asexual Phyllochaetopterus is accompanied by a corresponding decrease in the difference between the two regions in respect of the frequency of occurrence of motile individuals. Dividing cells were seen in all the hanging drops examined. The two cells resulting from division separate and become spherical before division takes place again, so that no chains or definite colonial groupings are formed. Both homogeneous and granular individuals of all sizes, except the smallest, are found dividing, and occasionally a pair is seen made up of one of each kind (fig. 6, PI. 26). No contractile vacuole, nucleus, or pyrenoid could be made out in the living organisms, nor, their colour being uniformly distributed, any definite chromatophore.

FILMS. Attempts were made to obtain further information on the structure of the green cells by making films from the suspensions obtained, as described in the preceding section, from each of the three species and submitting them to the action of reagents. Drops of the material were allowed to dry on slides smeared with glycerine-egg albumen and fixed for varying periods in the alcoholic fixative previously mentioned. The organisms were thereby more or less decolorized. Very little deformation of the cells seemed to result from this treatment. Chlor-zinc-iodine failed to indicate the presence of a cellulose cell-wall. The chaetopterin was quickly dissolved out of the cells, if they were not already completely decolorized, and the cells soon disintegrated. No blue or violet reaction could be detected in any completely decolorized cells. Iodine, either in alcoholic or sodium iodide solution, gave no starch reaction. In the case of Phyllochaetopterus and Leptochaetopterus inclusions staining a reddish brown 472 C. BERKELEY were seen in a few cells which may indicate the occasional presence of glycogen as a storage product. The decolorized cells could be recoloured with cytoplasmic stains. The structure in the granular individuals could best be brought out by staining with methylene blue, haematoxylin (Ehrlich's) or iodine. In some of the larger cells fine strands could be seen connecting the granules recalling the condition found in some of the unicellular Cyanophyceae (fig. 7, PI. 26). Several individuals differing in appearance from the usual type were found in films prepared from Phyllochaetopterus (fig. 8 a and b, PI. 26). In those shown at (a) the pigmented cell contents have concentrated into centres in a more or less regular manner. In those shown at (b) the formation of repro- ductive bodies seems to be involved. The cells contain smaller discrete bodies, but it has not been possible to count their number. Their expulsion from the parent cell has not been observed, but empty cells of large individuals are found in the films alongside a number of very small ones suggesting very strongly that the former has given rise to the latter. Neither granular cells nor this apparent spore formation has been seen in normal Mesochaetopterus, but when a portion of the abdomen of an individual of this species was kept in water in a closed vessel for some weeks, until the tissue had entirely decomposed, the green organisms not only remained intact, but were found, on examination in hanging drops and films, to be in an active state of division, and both granular forms and individuals showing suspected spore formation were present. In cells obtained from Phyllochaetopterus and Lep- tochaetopterus a more or less central, roughly circular body, staining well by prolonged treatment with Ehrlich's haematoxylin (diluted 1:5), is frequently present, which possibly may be a nucleus. Sudan III applied directly to films made from each of the three species confirmed the evidence obtained from the sections of material fixed with osmic acid fixatives that the green cells contain a considerable quantity of oil. As a rule the oily material GREEN BODIES OF CHAETOPTERIDAE 473 is extruded from the cells on drying and is shown in the films stained with Sudan III as a red field surrounding each group of green cells. Dividing cells, as described in the previous section, were found in all the films.

DISCUSSION. The colour of the intestinal wall of the three species of Chaetopteridae dealt with in this paper is, then, due to an infection of a green unicellular organism. Considering the very similar character of the green bodies in Chaetopterus variopedatus and of the pigment extracted from them, the explanation is almost certainly the same in this case also. Infec- tion has never been found absent in either of the three cases in- vestigated, and it takes place at a very early stage of the develop- ment of the annelids. This is not remarkable since the infective organism is always present in the contents of the gut and in the faecal masses, and the sexual products are carried out of the tubes by the same current of water as carries away the excreta. In a large majority of the cases in which the eggs of Meso- chaetopterus have been examined they have been found to carry the green infective organism externally. The organism remains normally in the palmella stage and increases only by fission ; it is found always in this condition in the older intestinal tissue. The flagellated form is found only in the recently generated segments and is clearly the agency by which the infection is carried into new areas. It can be assumed by individuals of various sizes under conditions favourable to swarming, as for instance in a suspension of the material teased out of the older tissue after standing some time, but, judging by the size of the organisms found in the larvae and in the recently budded segments, infection is normally brought about by the smaller individuals. There is no evidence on which a final opinion can be based as to whether the green organism is to be regarded as parasite or symbiont, and it is unlikely that the point can be decided with certainty unless the annelids can be bred under controlled 474 C. BERKELEY conditions, which hitherto has proved impossible. The epithelial cells of the gut are no doubt concerned in the disposal of organic waste material resulting from the digestive processes of the annelid. Probably, therefore, they afford an ideal environment for the development of the green organism, but there is no evidence that the annelid benefits in any way by the association. In the cases of Convoluta roscoffensis and Convoluta paradoxa, Keeble (11) has shown that, in addition to supply- ing carbohydrate and fat resulting from photosynthesis, the algae associated with these animals serve in lieu of excretory apparatus by absorbing their nitrogenous waste materials and are thus indispensable to their hosts. Photosynthesis is pre- cluded in the case of the chaetopterids owing to the conditions of total darkness in which the green organisms live, but it seems not unlikely that they play a similar part to the algae in Convo- luta by assisting in the disposal of the animals' waste products, and since they are always rich in oil, it is probable that the large quantity of that substance found in the intestinal cells results from their synthetic activity and serves a useful purpose in the annelid's metabolic processes. In this case the association must be regarded as symbiotic. On the assumption that the green organism absorbs the waste products of the worm, attempts have been made to grow it in liquid and on solid media containing uric acid and allied sub- stances as nitrogenous nutrients, but although a large number of media have been tried under a variety of conditions, no success has yet been attained. So far as can be judged without having the organisms isolated in pure culture they are very similar in all the three chaetopterids examined, though the slight morphological differences which have been mentioned may indicate varietal adaptions to each species. Without such cultures it may not be possible finally to determine the systematic position of the organism. It is evident that it is not an alga, as suggested by Brandes, if that term is used in its restricted sense to mean a definitely vegetable organ- ism adapted to photosynthesis, as in the cases of Zoochlo- rella in its various associations, the Chlamydomonas GHBBN BODIES OF CHAETOPTERIDAE 475 described by Keeble, and other similar instances, since it has neither cellulose, cell-wall, chromatophore, nor pyrenoid, and ap- pears to contain no algal pigment. It comes within the term if it is denned in its widest sense to comprise all the coloured Flagel- lata. According to Pascher's (12) classification of the pigmented flagellates, it must be included in the Chrysomonadae, though it fails in some respects to be a typical member of the group. Its characteristic of living normally in the palmella stage would then place it amongst the Chrysocapsinae, and it would come most nearly within the genus Chrysocapsa. The name Chrysocapsa chaetopteri is therefore tentatively sug- gested for the species. I wish to record my thanks to the Biological Board of Canada, and to Dr. Clemens, Director of the Pacific Biological Station, for the facilities placed at my disposal which have enabled me to carry out this investigation.

SUMMAKY. 1. The green bodies in the intestinal epithelial cells of three species of Chaetopteridae, each representing a different genus, have been examined. 2. They are very similar in the three cases and resemble those of Chaetopterus variopedatus. 8. They are frequently motile and flagellated when separated from the intestinal tissue. 4. They represent an infection of an organism which spreads in a flagellated condition. 5. The organism is a flagellate probably belonging to the family Chrysocapsinae. 6. The name Chrysocapsa chaetopteri is proposed for it. LITERATURE CITED. 1. Claparede, E.—' Struct. Annelides Sedentaires ', 1873. 2. Laffuie, J. J.—" fitude monograph, du Chaetopterus var. Chaetopterus variopedatus ", ' Arch, de Zool. Exp. et Gen.', Series 2, vol. 8, 1890. 3. Lankester, R.—" On the Green Pigment of the Intestinal Wall of the Annelid Chaetopterus ", ' Quart. Journ. Micr. Sci.', New Series, vol. 40, 1897. 476 C. BERKELEY 4. Brandes, G.—" DieUrsache der Griinfarbung des Darmes von Chaeto- pterus ", ' Zeit. f. Naturw.', vol. 70, 1898. 5. Brandt, C.—" tJber die Morphologische und Physiologische Bedeutung des Chlorophylls bei Thieren ", ' Archiv f. Anat. und Physiol., Abth. f. Physiologie', 1882. 6. Enders, E. E.—" A Study of the Life-history and Habits of Chaeto- pterus variopedatus," Renier et Claparede, ' Journ. of Morpholog.', vol. 20, no. 3, 1909. 7. Famintzin, A.—" Beitrage zur Symbiose von Algen und Thieren", ' Mem. Acad. Imp. Sci.', vol. 38, 1890. 8. Beijerinck, M. W.—" Culturversuche mit Chlorzellen, Lichtergonidien und anderen niederen Algen ", ' Bot. Zeit.', vol. 48, 1890. 9. Potts, F. A.—" Polychaeta from the north-east Pacific. The Chaeto- pteridae. With an Account of the Phenomenon of Asexual Reproduc- tion in Phyllochaetopterus and the Description of two new Species of Chaetopteridae from the Atlantic ", ' Proc. Zool. Soc. London ', 1914. 10. Berkeley, E.—" A New Genus of Chaetopteridae from the north-east Pacific, with some remarks on Allied Genera ", ibid., 1927. 11. Keeble, F.—' Plant-Animals. A Study in Symbiosis.' Camb. Univ. Press, 1912. 12. Pascher, A.—' Die Siisswasser-Flora Deutschlands, Osterreichs und det Schweiz ', Heft 2, Jean, 1913.

EXPLANATION OF PLATE 26. Fig. 1.—Transverse section of median region of intestine of Meso- chaetopterus taylori. Fig. 2.—A few cells of above highly magnified. Fig. 3.—A few cells from a transverse section of the median region of the intestine of Phyllochaetopterus prolifica. Fig. 4.—Cells from transverse section of intestine taken near the anal end of Mesochaetopterus taylori. Fig. 5.—Motile green organisms from Phyllochaetopterus pro- lifica. Fig. 6.—Stages in fission of green organisms from Phyllochaeto- pterus prolifica. Fig. 7.—Granular green organisms from Phyllochaetopterus prolifica. Fig. 8.—Specialized green organisms from Phyllochaetopterus prolifica.