The Metabolism of the Shore-Living Dinoflagellates: I.—Salinity and Carbon-Assimilation, in Amphidinium

Despite the abundance and economic importance of the dinoflagellates in the sea and on the shore, very little is known of the actual metabolic processes of even the commonest forms. West (1916), and Kofoid and Swezy (1921) have summarised our knowledge of the physiology of the group, but with the exception of some investigations on the luminescence of certain marine species, little or no experimental work bearing upon the group has been published. It is desirable, however, briefly to outline the present state of knowledge regarding dinoflagellate metabolism.

The dinoflagellates are in one respect unique, in that the general trend of the metabolic process is not yet definitely established, and that genera, and even species, in apparently the closest phylogenetic relationship, may exhibit the nutritive reactions typical, in the one case, of the holozoic, and in the other, of the prototrophic line of development. In addition, many species are saprozoic, at any rate facultatively, while it is even probable that a single individual, under the influence of different environments, may function as “animal” or “plant” (Zumstein, cited by Kofoid and Swezy, 1921, p. 92).

In many species of the Peridiniaceæ, both freshwater and marine, as well as in Amphidinium, and the simpler species of Gymnodinium and Gyrodinium among the naked forms, there are green, greenish-yellow, or brown chromatophores, in which chlorophyll, along with varying proportions of carotin and xanthophyll, appears to exercise a photosynthetic function. Other pigments—“phycopyrrin” and “peridinin”—have been alleged to exist (Schütt, 1890), but it is likely that further investigation will show these to be merely “chromophyll” complexes of chlorophyll with accessory pigments. It is noteworthy (Kofoid and Swezy, 1921, p. 51) that the presence of a photosynthetic mechanism is confined, in the main, to those species which are littoral or neritic in habitat, while forms not so provided, and presumably holozoic or saprozoic in their metabolism, are predominant in the eupelagic area, with a few representatives on harbour sands and muds, where suitable organic food-matter is available.

The holozoic forms frequently, but by no means always, possess reddish or violet pigments, either aggregated into granules or rodlets, or diffused throughout the cytoplasm. Various coloured inclusions, described as food-bodies, are frequently noted ; two genera, Polykrikos and Nematodinium, are provided with “nematocysts”; identifiable fragments, diatoms, and even other dinoflagellates, have been seen within the cell, but in no case has actual ingestion of solid particles ever been observed. Schilling (cited by Kofoid and Swezy, 1921, p. 312) has described the formation of amoeboid pseudopodia, to aid the ingestion of food-particles, in Gyro-dinium hyalinum, but in the case of the peridinians, the rigid plate-armour seems to be an effectual barrier against any form of ingestion. The so-called pusule apparatus is, no doubt, the digestive organelle in the holozoic and saprozoic species, and drops of oily reserve-matter are to be found in close association with it. Starch also occurs as a reserve product, and is not confined to the green, and presumably prototrophic, forms—it is found in the chromatophores or in special amyloplasts.

A remarkable metabolic phenomenon, closely paralleled in the Myxophyceæ, is to be seen in the occasional development, in enormous quantity, of some of the species normally occurring in the marine and freshwater plankton. In fresh water, Ceratium hirundinella is one of several forms which may give rise to the “water-bloom” sometimes seen in lakes and reservoirs, while in the sea, especially in warm-temperate regions, members of the genera Ceratium, Gonyaulax, Peri-dinium, and Prorocentrum may become so abundant as to discolour the water and give rise to malodorous products of metabolism and decay which are toxic to some fishes and bottom-living animals.

Several of the most highly specialised genera in the group are distinguished by the possession of an ocellus. This, while not in itself of direct metabolic significance, has a bearing upon conditions of nutrition, and the forms so provided are apparently holozoic. Several simpler types, not provided with any specialised receptive organelle, are still markedly sensitive to light, and react positively to illumination of moderate intensity. The vertical migrations of Amphidinium on the beach sands at Port Erin, first noted by Herdman, (1911 a), are almost certainly determined by the alternating rhythms of tide and light.

Finally, a number of dinoflagellates are luminescent. Among them, Ceratium, Noctiluca, and “Pyrocystis” are responsible for the more marked displays of phosphorescence 4’5 in temperate seas. This striking phenomenon has naturally attracted much attention, and a considerable literature has grown up around it, which cannot even be summarised here (videHarvey, 1920).

It is evident, from the foregoing summary, that more extended field-observations and much experimental work are needed before any clear understanding of the metabolism of the dinoflagellates can be obtained. At the same time, the group is too large, and the conditions of existence too diverse, to render possible anything like a complete study, and the present investigation is limited, therefore, to the shore-living forms.

Experimental work has aimed, so far, at determining the influence of the separate factors, light, temperature, salinity, tidal rhythm, pH, organic matter, and associated living organisms, upon the metabolic activities of Amphidinium herdmani, Kofoid. (This is the species most abundant on the Port Erin beach, originally noticed there by Herdman, and described by him (1911) and by Laurie (1914) as Amphidinium operculatum, Clap, and Lach.). Numerous field-observations are recorded, in the literature quoted, as to the occurrence and periodicity of Amphidinium on the sandy beaches at Port Erin, Hoylake, and elsewhere, but it is clear, from the somewhat conflicting evidence, that the responses have been evoked, for the most part, by complex causes. The need is thus apparent for determining the effect of separate factors, under controlled laboratory conditions. Numerous difficulties have been met with in the course of the work, and the observations bearing on the influence of salinity are alone in a sufficiently forward state for publication.

The methods adopted have been determined largely by the rather special requirements of the organism. It has not been found possible, up to the present, to obtain successful cultures of Amphidinium lasting more than a few days. Various methods and media have been employed, but sooner or later the Amphidinia degenerate, and large numbers of ciliate infusorians appear in the liquid, obscuring any effects due to the Amphidinia themselves ; in consequence, it has been necessary to rely on fresh supplies of the living Amphidinia scraped from the surface sand of the beach. While this method has ensured the fullest vigour of the organisms under observation, it has the disadvantage that the periodic fluctuations in abundance, under natural conditions, seem rarely to coincide with laboratory requirements, and that only occasionally can scrapings be obtained in which organisms other than Amphidinium herdmani are present in negligible quantity.

The scrapings, usually several hundred c.c. in volume, were taken from noticeably discoloured patches of the sand, at about half-tide level or somewhat higher, over which one or other of the freshwater streamlets which cross the beach, was trickling. At the same time, by scooping a slight hollow in the sand, it was possible to obtain a sample of the interstitial brackish water, of which, the temperature and specific gravity having been noted, the salinity was readily deduced by reference to Knudsen’s Tables. The sand-scrapings, containing the Amphidinia, were then shaken into two or three litres of sea-water, appropriately diluted, and contained in a large, shallow, enamel vessel. After standing for several hours, in a well-lighted window, out of direct sunshine, the Amphidinia were found massed at the surface and on the sides of the vessel, and could be pipetted off, with the smallest possible volume of water. The suspension thus obtained was of a deep olive-brown colour, and was found to be practically free from diatoms and other “foreign” organisms. For the present observations, no attempt was made to free the suspension from bacteria ; it will suffice to mention that no trace of opalescence or putridity was noted, at any stage, in any of the recorded experimental series.

This was regarded as the “standard suspension” from which appropriate dilutions were made in any single experiment. Short of an actual count of the narcotised organisms, in a hæmacytometer, which was not available, it is difficult to see how the strength of such a suspension could be estimated, and in any case the growth and division of the organisms during the experimental period is a factor to be reckoned with. As a result, the determined values of metabolic activity are relative only—comparable with other members of the same series, but not with other experiments or other organisms. As the roughest approximation, it may be noted that 10 c.c. of the standard suspensions usually employed gave, on centrifuging, 0.03 to 0.05 c.c. of “solid” Amphidinia ; assuming that the volume of a single Amphidinium is 4 × 10⁸ cubic microns, the standard suspensions contained about 100,000 individuals per c.c. ; this number was reduced by dilution in the actual experiments.

The arrangements varied somewhat in the different series, but in every case the method adopted to determine the net metabolism was that described by the author in a previous paper in this Journal (1924), where reference is made to the precautions necessary, correction for “salt-error,” and other conditions involved in the pH method.

At the outset, it was necessary to establish that the metabolic activity of a large number of Amphidinia, confined in a small volume of water, did not produce any appreciable change in the concentration of excess base. Two resistance-glass -flasks, each containing 300 c.c. of sea water diluted with an equal volume of distilled water (i.e. at a salinity of about 17 per mille), were allowed to stand for several days in a N. window, one (A) being inoculated with 5 c.c. of a fairly rich suspension of Amphidinium herdmani, and the other (B) allowed to stand as a control. Samples were removed periodically, for pH and excess base determination, with the following result :—

Thus it is clear that even vigorous carbon assimilation is not accompanied by any demand for basic materials.

The following experiment was designed to determine, in a preliminary way, the range and amplitude of pH variation brought about, in water of various degrees of salinity, by the photosynthetic activity of suspensions of Amphidinium. Into each of five conical resistance-glass flasks was placed a dilute suspension of Amphidinium, made by adding 10 c.c. of a standard suspension to 200 c.c. of sea water, variously diluted, as shown in the table below :—

The turbidity produced by the suspended organisms was not such as to interfere with the indicatometric measurement of pH. 10 c.c. were immediately withdrawn from each, for pH determination, and the flasks were then plugged with cotton-wool and set outside in full daylight. Samples were removed at intervals throughout several days ; the contents of the flask were gently rotated before removal of the sample, but not shaken in a manner likely to cause extensive CO₂-exchange between the liquid and the air in the flask. Samples removed during the hours of darkness, when immediate pH-determination was not possible, were preserved with a drop of pure toluene, and securely corked ; this treatment led to no appreciable change of pH after an interval of ten to twelve hours. Thymol blue was used as indicator for series A, B, C, and D, and phenol red for series E ; the pH values recorded are in all cases corrected for salt-error. The excess base was determined, by the method described in the previous paper (Bruce, 1924), on 100 c.c. of the media used, in each case. The temperature, during the course of the experiment, varied between 10° and 12°C. The results obtained, expressed directly as pH variations against time, are set-out in Table I. and Fig. 1.

Conversion of the pH values into amounts of CO, assimilated would serve no useful purpose, since no special precautions were taken to ensure CO₂-equilibrium at the outset, nor to prevent access or loss of the gas during the course of the experiment. The results, however, are comparable among themselves, and bring out several points of interest.

The diurnal periodicity is noteworthy, in that the assimilatory maximum appears to coincide, not with the maximal illumination, but with a point of considerably less intensity, at about 5 p.m. The amplitude of the daily pH variation apparently increases with the age of the culture, but this may be due either to multiplication of the organisms, or to concentration of the suspension brought about by the adherence to the glass of some of the Amphidinia, and consequent removal in sampling of a non-aliquot fraction of the whole.

Of more immediate interest, however, are the differences associated with varying salinity. It is at once obvious, from inspection of the curves in Fig. 1, that the organisms are photosynthetically active in solutions A, B, C, and D, i.e. in all salinities from that of sea water down to one quarter of that strength, and over a range of excess base from 25.0 to 7.75. In fresh water, however, of low excess base, assimilation, as far as may be judged from pH increase, seems to be greatly depressed, and this despite the fact that the low buffer value of fresh water should lead to a greater change of pH for a given gain or loss of carbon dioxide. For the reasons stated above, it must not be assumed that the greater rise of pH after a given period of illumination, noted in the suspensions of lower salinity, is necessarily indicative of a greater degree of photosynthesis though, as will be shown in the following experiment, salinity (or possibly its covariant, excess base) has a very definite influence, quite apart from CO₂-tension, on the rate of carbon assimilation.

In the following experiment, various modifications, suggested by the foregoing, were adopted. Hard-glass test-tubes, each of 25 cc. capacity, were filled to the top with active suspensions of Amphidinium herdmani, all of the same strength, and prepared as before, but on this occasion in nine different dilutions of sea water. Several tubes were filled with each of the nine dilutions, paraffined corks were then inserted, and the tubes, supported in test-tube racks, placed in diffused light out of doors, care being taken to secure equal exposure for all the tubes. Prior to the addition of the standard suspension, the sea -and fresh-water mixtures had been violently shaken, for at least ten minutes, with a large volume of air, with a view to ensuring equilibrium with the atmospheric CO₂-tension (at 762.5 mm. and 14^o C.), and initial equality as between the different series. It was found, later, that equilibrium was not quite attained under these conditions, but the CO₂-tension gradient between the successive samples was such as to diminish, rather than exaggerate, the differences in rate of carbon assimilation, as shown by the pH values attained.

Excess base and salinity were carefully determined on the fresh water and sea water used, and the data relative to each of the mixtures are shown below :—

In view of the limited volume of water, and consequently of carbon dioxide, available for photosynthesis, it was regarded as desirable that the/H change should be as small as possible, compatible with accuracy in measurement. A tube, once opened for the removal of the 10 c.c. required for pH determination, was not further used, and it is believed that the readings obtained were not influenced by loss or gain of carbon dioxide other than that due to the gaseous exchanges of the suspended organisms. Determinations of the pH were made, initially (at 3.45 p.m.), and again after an interval of three hours’ exposure to dull diffuse light, using cresol red as indicator. A further series was removed (at 4.30 a.m.) when it was supposed that the respiratory activity, proceeding in darkness, would have attained its maximal effect. In this case the tubes were momentarily opened for the addition of a drop of toluene, and were then corked again, until pH determination was possible, in daylight, five hours later. The three series of readings, corrected in all cases for salt-error, are given in Table II, and in Fig. 2 are plotted against salinity.

Over a certain range of salinity, roughly from that of normal sea water down to half that strength, the pH values attained after three hours, under the conditions of illumination prevailing in the experiment, are lower than initially, while in water containing less than 18 parts of salts per 1000, an increase of pH is noted, the maximum difference of 0.2 pH lying at about 6 per mille salinity. These relations are clearly evident in Fig. 3, where the differences of pH, positive and negative, at the expiration of three hours (as read from the smoothed curves), are plotted against the corresponding salinities, the pH values being read from the ordinate on the left.

In order to ascertain the influence of salinity on the actual metabolic activity, the total CO₂-content, corresponding to each pH and excess base value, was read from the curves given in the author’s previous paper (1924). The results so obtained, plotted as the lower curve, and read from the right ordinate, in Fig. 3, indicate very clearly the trend of carbon assimilation in solutions of progressively increasing salinity. The net loss of total carbon dioxide,” due to fixation presumably as carbohydrate, practically nil after three hours in fresh water, reaches a maximum at from 4 to 8 per mille salinity, thenceforward falling until, at 18 per mille salinity, assimilatory activity can no longer cope with the respiratory output of carbon dioxide, and at the highest naturally occurring salinities, there is a marked increase in the total CO₂-content.

It is evident that if the physical conditions—high salinity and feeble illumination—leading to such a result, obtained for any length of time, a heterotrophic type of nutrition would alone meet the reproductive and energy requirements of the organism. Facultative changes in metabolic activity, as mentioned in the introductory remarks, are believed to take place in several genera of the dinoflagellates—the results now put forward define the conditions under which such a change, in Amphidinium, might be looked for, and this possibility is being further pursued. It must not be overlooked that the recorded differences of CO₂-content are net values — the resultant of CO₂-decrease due to photosynthesis, and CO₂-increase due to respiration. If analogy with the typical green plant is permissible, the rate of carbon assimilation, in bright light, far exceeds the simultaneous respiratory output of carbon dioxide. The values determined as at 4.30 a.m., and plotted as the lowest of the three curves in Fig. 2, while affording a rough indication of the respiratory activity, are not strictly comparable among themselves, since each set of organisms entered upon the period of darkness at a different oxygen -and carbon dioxide-tension. Further work, however, is in progress, as a result of which it is hoped to determine the extent of the purely respiratory exchange, and the influence, if any, upon it, of salinity and other factors.

In the meanwhile, the net results, as they stand, find remarkable confirmation in the distribution of Amphidinium herdmani on the sandy beach at Port Erin and elsewhere. As previously noted, the discoloured patches are practically confined to those areas, at about half-tide level, which arc subjected, at every ebb of the tide, to dilution and washing by fresh water. When scrapings have been collected from these areas, and on other occasions, samples of the interstitial water, oozing into a hollow made in the sand, have been examined for salinity. The values obtained, together with an indication of the dominant organism, are here given.

It is evident that an abundance of Amphidinium herdmani on the seashore is associated with a salinity-range close to that shown by experiment to be optimal for assimilatory activity.

The biological and economic importance of the marine Dino-flagellata, as well as the unique taxonomic position of the group, call for a thorough investigation of their metabolic relations.

As a preliminary to the wider problem presented by the planktonic genera, the shore-living forms are being studied with a view to determining their metabolic processes and the physical and biological factors which influence their relation to the general nexus of living forms in the tidal area.

The present report deals with salinity as a determining factor in the distribution and photosynthetic activity of Amphidinium herdmani, Kofoid, one of the more abundant dinoflagellates in the Port Erin area.

It is shown, experimentally, that the organisms, suspended in water and exposed to diffuse daylight, are capable of assimilating free carbon dioxide, at all salinities, from that of normal sea-water down to practically fresh-water. The rate of assimilation, however, is low when the salinity lies outside an optimal range (4 to 8 per mille), and it is possible that under these conditions Amphidinium may function, facultatively, as a heterotrophic organism.

Determinations made upon samples of interstitial water, taken on the beach, demonstrate that salinity is a factor—there are doubtless others—in the distribution of Amphidinium, and that the strongly discoloured areas, where it is present in great profusion, are characterised by salinities lying within the optimal range indicated above.