ABSTRACT
The oxygen consumption of S. ekmani at 5° C is 0-06 /μl./mg./hr.
Phenylalanine and glycine are concentrated by S. ekmani. The concentration factor reaches a maximum after 30 min. in animals removed from their tubes. In animals inside their tubes, the rate of uptake is limited by the rate of diffusion through the walls of the tube. The phenylalanine does not move appreciably into the alcohol-insoluble extract of the animals over a period of 1 hr.
Protein is taken up by S. ekmani when animals are removed from their tubes. Uptake is slower than uptake of amino acids, and may involve a different mechanism.
Autoradiography using S. mergophorum shows that phenylalanine is not adsorbed on the cuticle. It is found especially in secretory cells, within which it is localized over rough endoplasmic reticulum, Golgi regions and secretion spherules.
The site and mechanism of uptake of organic molecules are discussed; and the types of molecules absorbed, together with the significance these may have in the overall metabolism, are considered.
INTRODUCTION
The phylum Pogonophora consists of marine tube-dwelling worms living mainly in areas of soft mud, at temperatures usually below 10° C, and therefore often at considerable depths. One of the most interesting features of the phylum is the lack of any conventional gut or digestive system (Ivanov, 1963). Ivanov has suggested that the Pogonophora are filter feeders, utilizing their anteriorly placed tentacles and some mechanism of external digestion ; but the scarcity of cilia in some forms, and the apparent lack of any cells which might produce digestive enzymes (Gupta, Little & Philip, 1966), as well as an apparent lack of proteolytic enzymes demonstrable by histochemical methods (Southward & Southward, 1966) all cast doubt on this hypothesis. Jägersten (1957) revived the theory of Pütter (1909) that dissolved organic substances might be taken up directly, and preliminary experiments by Little & Gupta (1968), and investigations by Southward & Southward (1968) have provided evidence that amino acids and even proteins can be taken into the cells. In fact the uptake of dissolved organic substances by various marine invertebrates has been demonstrated by a number of workers, although in all these cases there has been doubt as to the significance of the uptake in overall metabolism (e.g. Johannes, Coward & Webb, 1969). Uptake has been shown for polychaetes by Stephens & Schinske (1961), Stephens (1963, 1964), Chapman & Taylor (1968) and Taylor (1969); for corals by Stephens (1962); and for echinoderms by Stephens & Virkar (1966) and Ferguson (1964, 1967).
The present paper provides information concerning the uptake of amino acids and proteins from sea water by certain Pogonophora.
MATERIAL AND METHODS
Pogonophora were collected in the Raunefjord and the Korsfjord, near the Biological Station of the University of Bergen, Espegrend, Blomsterdalen, Norway. An Agassiz trawl was used at depths of 240 m. in the Raunefjord and 680 m. in the Korsfjord. The bottom temperature is 6–7° C. Pogonophora caught in the chains and in the net were quickly transferred to sea water at 5° C, and returned to the laboratory of the Biological Station, where they were kept in 2 1. containers at the same temperature. Survival was excellent for periods of 2–3 weeks, but all experiments were carried out within 1 week of collection. Animals used were all complete as far as the annulus, but only a few retained the postannular region. The species used for almost all the physiological work was Siboglinum ekmani Jägersten, but some measurements of rate of respiration were made with S. ftordicum.
The material for autoradiography was obtained from Miami, U.S.A. Siboglinum mergophorum were dredged from 200 m. off Miami Beach, Florida, where the bottom temperature is about 8° C. Animals were removed from the dredge, placed in sea water at 8° C, transported back to the Institute of Marine Sciences, University of Miami, and maintained in running sea water at 8° C until being used the next day.
Rate of respiration
To obtain a crude measurement of the rate of oxygen uptake groups of animals with tubes cleaned, rinsed in ‘Millipore’-filtered sea water and with excess tube trimmed away, were placed in ground-glass stoppered bottles of 40–70 ml. capacity also containing ‘Millipore’-filtered sea water (pore size 0·45 μ). The bottles were placed in the dark at 5° C, and the oxygen content of the sea water was measured at the start of the experiments and after 52–110 hr., using the Alsterberg (azide) modification of the Winkler method for dissolved oxygen. Readings were also taken for simultaneous blanks.
At the end of each experiment the worms were removed from their tubes using fine forceps and needles, and were weighed on a Mettler B6 balance (accuracy 0·02 mg.). Weighings were made at intervals of 30 sec., and the initial wet weight was established by extrapolation back to zero time. This measurement of wet weight is liable to incorporate a large degree of inaccuracy due to the unknown quantity of water remaining attached to the animals; and it may account for some of the variation in Text-fig. 1. Weights taken in this way will be maximum estimates, but will produce similar errors in the estimation of rate of respiration and of rate of uptake of amino acids.
Uptake of amino acids
Pogonophora were cleaned as above, and individuals were placed, either in their tubes or after these had been removed, into 1–10 ml. of solutions of 14C-phenylalanine or 14C-glycine (Radiochemical Centre, Amersham) in ‘Millipore’-filtered sea water. They were kept in the dark at 5° C for periods of 5–60 min. After this time they were removed from their tubes if necessary, washed for 20 sec. in clean cold sea water, weighed as above, and then extracted for 3 hr. in 100 μl. of 80% ethyl alcohol. 50 or 75 μl. of this extract was then transferred to a planchet, as were samples of the sea water before and after the experiment. The animal was then rinsed in distilled water, and macerated on a planchet. Samples were dried down under an infra-red lamp, and 14C was counted using a Frieseke and Hoepfner gas-flow counter. In general the time for 1000 counts was taken 2–3 times, but for active samples 10,000 counts were taken. Background was approximately 14 counts/min.
Uptake of protein
Denatured 14C-labelled Chlorella protein, 100 µc., was dispersed in 200 ml. ‘Millipore’-filtered sea water, to which had been added 2 mg.% penicillin G and 2 mg. % streptomycin sulphate to prevent bacterial and fungal action. The degree of uniformity of dispersion of the protein can be judged from the s.E. of samples taken before and after experiments: 1570 counts/min./10μl. ± 123·5 (20 samples). Pogonophora were placed individually in 10 ml. of this protein suspension for periods of 10 or 60 min., and were then rinsed as above, and macerated on planchets. After drying down they were counted for 14C directly.
In all these experiments, corrections have been made for self-absorption and for background.
Permeability of the tubes to phenylalanine
Empty tubes were placed in a damp chamber, and 2 × 10−4M 14C-phenylalanine in ‘Millipore’-filtered sea water was injected from a microburette. The ends were sealed with dental wax, and the tubes were immersed in small glass tubes containing 500 μl. of sea water, at 5° C. This was stirred by blowing air in through a fine glass tube. Samples of sea water, 10 μl., were taken at intervals (from 1 min. to 6 hr. after immersion), dried down on planchets, and counted for 14C on a Nuclear-Chicago gas-flow counter. The figures obtained were adjusted for background, self-absorption and for volume changes incurred by removal of samples.
The dimensions of the tubes, which were approximately 5 cm. long and 0·01 cm. in diameter, with a volume of 0·5–1·0 μI., were measured using a binocular microscope (magnification × 200).
From the efflux curves obtained, the maximum reading was treated as a base line, and the slope of the curve was determined half-way between this and zero counts, i.e. at the point where half the amino acid had moved out of the tube. The concentration inside the tube was therefore 1 × 10−4M. The flux,/, was calculated from this slope. The permeability coefficient, P, was then calculated from
where A is the area of the tube in cm.2, estimated using the average of internal and external radii, and C is the concentration in M/cm.3. The assumption was made that P would be similar at lower concentrations than 10−7M, and the flux at 2 × 10−7M was calculated.
Autoradiography
Specimens of Siboglinum mergophorum from Miami, Florida, still enclosed in their tubes, were placed in 2 XIO−5M 3H-phenylalanine (S.A. 5·0 Ci/mM) in ‘Millipore’-filtered sea water. The final concentration of the label was 100 μCi/ml. sea water (35 %o S) at 8° C. Specimens were removed either after 1 hr. or after 20 hr. To process the material for microscopy, animals were removed from their tubes, rinsed in several changes of filtered sea water, and fixed for 2 hr. in 4 % glutaraldehyde (Biological grade) in 0·1 M sodium cacodylate buffer at pH 7·4 with added sucrose to give a final measured concentration of 1100 m-osmoles/1. Fixed animals were then cut into suitably small pieces, washed thoroughly in several changes of sucrose containing cacodylate buffer (1100 m-osmoles/1., overnight at 8° C), post-fixed in buffered OsO4, dehydrated and embedded in ‘Araldite’ (Gupta et al. 1966; Gupta & Little, 1969a).
For light-microscopic autoradiography, 0·5 μ thick sections of the ‘Araldite’ blocks from various regions of the animals were mounted on gelatine-subbed glass slides and coated with Ilford L4 liquid nuclear track emulsion. The details of such a procedure are given by Rogers (1967). All the requisite controls for chemography and other artifacts (Rogers, 1967) were used. Autoradiograms were exposed from 2 days to 10 weeks, developed in Kodak D19b or D170 for 3–5 min. at 20° C, fixed in 25% sodium thiosulphate, washed and dried. They were examined either unstained by phase-contrast and dark-ground microscopy, or stained briefly in 0·5 % toluidine blue through the gelatine.
For electron microscopy, 500–1000 Å thick sections were mounted on glass slides coated with celloidin films. Sections were stained with uranyl acetate and lead citrate (Gupta et al. 1966), dried and covered with a thin layer of evaporated carbon (Salpeter & Bachmann, 1964). Slides were then coated with a tightly packed monolayer of either Ilford L4 (purple interference colour) or Agfa-Gevaert NUC 307 (pale gold interference colour) nuclear track emulsions, using a Kopriwa-type semi-automatic coating machine (Kopriwa, 1967a). Under our laboratory conditions, the dilution needed was 2:3 for Ilford L4 and 3:2 for Gevaert NUC 307; both emulsions being used at 35° C and with a withdrawal speed on the machine of 48 mm./min. (cf. Kopriwa, 1967 a). Thoroughly dried preparations were stored over silica gel in light-tight boxes at 4° C for 6–12 weeks. Autoradiograms were processed in an appropriate developer for each emulsion (Kopriwa, 1967b), fixed and washed. The section-bearing areas of the celloidin films were floated on water and mounted on naked copper grids for electron microscopy (Salpeter & Bachmann, 1964; Rogers, 1967). When thoroughly dry, grids were examined, without any further treatment, in a Philips EM 200 electron microscope fitted with a ‘cold-finger’ anticontamination device.
RESULTS
Oxygen consumption
Since part of the object of this work was to try to estimate the significance of the direct uptake of organic substances in the metabolism of Pogonophora, crude measurements were made of the rate of uptake of oxygen, using groups of 10–14 animals for each determination. Animals were used in their tubes, and these often have protozoa attached, or harbour bacteria and fungi. In two series of experiments animals were placed for 2 hr. in either 2 mg. % penicillin G and 2 mg. % streptomycin sulphate, or in 0·01 mg. % chloromycetin, before transference to the closed bottles for measurement of oxygen uptake. Results are shown in Table 1, and suggest that neither bacteria nor fungi on the tubes contribute significantly to the rate of uptake of oxygen by the Pogonophora.
These results are of the same order as those found by Manwell, Southward & Southward (1966) for the larger form Siboglinum atlanticum. For example, a single determination made by these workers soon after collection of the specimen gave a result of 0·1203 ml. O2/hr./gm. wet weight, at a temperature of 15° C, as compared to a value of 0·060 ml. O2/hr./gm. for S. ekmani at 5° C.
Uptake of amino acids
Two amino acids were employed : uniformly-labelled 14C-glycine, and 14C-phenyl-alanine. Most experiments were carried out with the latter because it is available at a higher specific activity than glycine. Initially, the relationship between weight and rate of uptake was briefly investigated. With an experiment time of 10 min., and using 2 × 10−7M phenylalanine, the amount of radioactivity in 75 μl. of alcohol extract was plotted against the wet weight of animals extracted (Text-fig. 1). Since the scatter was very great, and since most of the animals used in subsequent experiments were between 0·3 and 0·6 mg., adjustment for variation in weight was made by dividing the radioactivity of the extract by the weight of the animal. This figure was then expressed as counts/min./mg. (wet wt.) of animal, and was compared directly with the figure for counts/min.//μl. sea water, to give a ‘concentration factor’. The true relationship of weight to amino acid taken up is probably logarithmic but in the present case there is too much scatter to decide this, and the linear relation in fact approximates to the logarithmic one over the range of weights used.
The comparison between animals in their tubes and those taken out may be criticized on the basis that damage may occur during the extraction from the tube ; although no visible damage occurred. Similarly, all the experiments may be criticized because few of the animals were complete ; although those that lack the postannular region appear to seal off the break behind the annulus. Against such criticism, it may be pointed out that since the animals actually concentrate amino acids in a relatively short time, the importance of diffusion through wounds and damaged areas, which would only occur down concentration gradients, must be negligible.
(a) Phenylalanine
A concentration of 2 × 10−7M was employed, as this represents 36 μg./l. (at the S.A. used), this value being of the same order as the observed total concentration of amino acids in sea water (Degens, Reuter & Shaw, 1964; Chau & Riley, 1966). Results obtained over a time of 60 min. are shown in Text-fig. 2 for 5. ekmani, both when in their tubes and when removed from their tubes. It is evident that with the animals removed from their tubes a high uptake rate occurs up to 20–30 min., and that this falls off after longer periods. The fall-off is presumably due to a balance being reached between uptake and loss via diffusion, metabolic breakdown and secretion. The rate of uptake by animals still in their tubes is much slower, and over 1 hr. the curve appears to be linear; the preliminary results of Little & Gupta (1968) show that concentrations comparable to those produced by animals without tubes can be reached over periods of 13–20 hr.
Two further variations in the treatment with phenylalanine were used. First, animals out of their tubes were exposed for 10 min. to 2 × 10−7M phenylalanine with 2 mg. % penicillin G and 2 mg. % streptomycin sulphate to stop bacterial action. Second, the concentration of phenylalanine was increased to 2 × 10−6 M, and observations were made of uptake after 10 min. These values are compared with those for 10 min. experiments at 2 × 10−7 M with no added antibiotics, in Table 2. There appears to be no effect on uptake when antibiotics are added, suggesting that bacteria are not involved in the uptake process. With a tenfold increase in concentration, concentration factors are similar to those at 2 × 10−7M.
Measurements of the amount of radioactivity incorporated into the alcohol-insoluble fraction during all these experiments showed that only 3–4% of the 14C taken up was incorporated into this fraction. There appeared to be no significant percentage increase in the alcohol-insoluble fraction with increased time of exposure to amino acids. The figures for 5. ekmani out of their tubes are given in Table 3, and are of the same order as those given by Stephens & Virkar (1966) for the brittle star Ophiactis.
(b) Glycine
A concentration of 6 × 10−7M (or 46·8 μg./l. at the S.A. employed) was used. Uptake after 10 and 60 min. is shown in Table 4. The results are comparable to those obtained with phenylalanine, except that the uptake after 10 min. by animals inside their tubes is higher for glycine than for phenylalanine: for phenylalanine, the concentration factor is 1·15 ± 0·477 (5)> and for glycine it is 2·98 ± 0·562 (5) (t = 2·453, P = 0·05 – 0·02).
Measurements of the alcohol-insoluble fraction were similar to those obtained with phenylalanine (Table 3), and suggest that very little of the accumulated carbon is incorporated into protein over the short time-period of 1 hr.
(c) Permeability of the tubes to amino acids
The figures for the flux of phenylalanine at 2 × 10−7 M are given in Table 5. The length of tube used was approximately 5 cm., which is of the same order as that in-habitated by 2–3 animals, which would weigh about 1 mg. The figures for fluxes can therefore be directly compared with the figures for uptake of phenylalanine by animals within their tubes. The slope of the relevant line in Text-fig. 2 corresponds to an uptake rate of 0–00023 μg/mg. animal/hr. Although the figures for flux rates are very variable, they are of the same order as this, suggesting that the rate of passage through the wall of the tube is the main limiting factor controlling uptake; and that the passage of amino acid in from the end of the tube is not greatly significant. This hypothesis gains support from some brief experiments using dyes. Small amounts of methylene blue were injected into the lumen of tubes containing living S. ekmani ; but although the animals moved slowly up and down their tubes the dye had not moved appreciably after 12 hr., showing that no ciliary current moves into or out of the aperture of the tube.
When the figures for protein are considered (see next section), it is obvious that no protein is penetrating the tube walls. The tube is, in fact, acting as a filter, allowing through only small molecules. This is a strikingly similar phenomenon to that shown by the rectal intima of the locust Schistocerca (Philips & Dockrill, 1968), which allows the passage of small molecules, but prevents the passage of inulin and proteins. The comparison is even more interesting when it is noted that both membranes are chitinous, and that the permeability coefficients of the rectal intima for small molecules are similar to that for phenylalanine of pogonophoran tubes.
Details of the structure of pogonophoran tubes will be given by Gupta & Little (1969c).
Uptake of protein
In order to test the ability of S. ekmani to take up protein, individual animals were placed in a suspension of denatured Chlorella protein (Radiochemical Centre, Amersham; ‘freed of water-soluble compounds by molecular filtration’), to which was added 2 mg. % penicillin G and 2 mg. % streptomycin sulphate to prevent bacterial breakdown. The concentration used was 500μC./I., or 1000 μg./L, ± 5%. The results nave been calculated in a similar way to those for amino acids, but although the protein is in suspension, the dispersion is not perfect (see Methods).
Table 6 shows the results, from which it is obvious that the situation is somewhat different from that obtained with amino acids. In 10 min. animals out of their tubes take up very little protein; but after 60 min. a concentration factor of 11·59 is reached. This suggests that whatever process is responsible for the uptake of protein it takes some time to build up, unlike the process concerned with the uptake of amino acids, which certainly commences within 5 min, of immersion of the animal in an amino acid solution. Animals inside their tubes take up virtually no protein, even after 60 min., suggesting that the protein is too large to pass through the walls of the tube, and that it is not drawn in through the openings at the ends of the tube.
Autoradiography
The technique of autoradiography employed here will demonstrate only that portion of the total sH-phenylalanine taken up by the animal which is either bound or has been incorporated into molecules that survive subsequent processing of the tissue. This fraction will probably be similar to the alcohol-insoluble fraction in the data given above, and will therefore probably not constitute more than 5 % of the total uptake, at least over the short time range of 1 hr. The technique, therefore, does not indicate the sites of uptake but the sites of metabolic incorporation into the tissue and cell components. In the final autoradiographs such sites of incorporation are indicated by the presence of silver grains and the accuracy of this localization is limited by the autoradio-graphic resolution. With the methods employed here, this resolution is expected to be better than 2000 Å with Ilford L4 emulsion, and better than 1000 Å with Agfa-Gevaert NUC 307 emulsion (Bachmann, Salpeter & Salpeter, 1968).
Light-microscope autoradiographs provide information on gross localization of the label over different parts of the animal, and preliminary results from such preparations have already been published (Little & Gupta, 1968; Southward & Southward, 1968). In such preparations, silver grains are formed over the tentacle as well as all over the main body of the animal as far back as the annulus. No material was available from the region posterior to the annulus. High concentrations of grains occur over areas where high metabolic or synthetic activity is expected, such as the base of the tentacles and the cephalic lobe, in the forepart (Little & Gupta, 1968), and in the region of the metameric papillae and the ovary in the trunk. Even at high magnifications the surface of the animals, the cuticle, and the ciliary band do not show any significant binding of the label; the grain density over these regions being no higher than the background level.
High-resolution autoradiographs examined by electron microscopy confirm the general pattern of incorporation of the label and provide further details of a differential distribution of autoradiographic grains over various tissues and over different types of epidermal cells (see Southward & Southward, 1966, for a histochemical description of these cells). Tables 7 and 8 summarize the distribution patterns of autoradiographic grains over different tissues and cell types after 1 hr. and 20 hr. of radioactive in-corporation respectively. The results are expressed both as grain counts/100 μ2 of the tissue in sections and as a ratio of the percentages of total grains counted and of total area occupied by a particular tissue in all the electron autoradiographs examined in the present study. This last value permits a direct comparison of the relative incorporation Bf the radioactivity by different tissues and cell types irrespective of the emulsion used.
It clearly emerges from this data that in S. mergophorum there is no significant binding of the label in the cuticle and associated surface material even after a 20 hr. exposure to 3H-phenylalanine (Pl. 1, Tables 7, 8). In specimens kept in the label for 1 hr. (Table 7) autoradiographic grains are formed over all other tissues including the blood but the maximum concentration occurs in the epidermal cells which in this case include the pinnule cells of the tentacles. A separate analysis of the grain distribution over the pinnules did not reveal any significant difference in grain/area ratio from the rest of the epidermal cells.
In specimens kept in the label for 20 hr. the general pattern of grain distribution is maintained (Table 8). Furthermore, different cell types in the epidermis show a wide range of radioactive incorporation. A maximum concentration of grains is found over the cells which by their distribution in the animal are identified as ‘protein secreting cells’ in the metameric papillae of the metasome (Plate 2). These cells probably correspond to the ‘white protein cells’ described in S. atlanticum and histochemically analysed by Southward & Southward (1966) as producing a tyrosine-rich protein secretion. The lowest grain/area ratio is found over the secretion contained in the lumina of the pyriform glands which is believed to be the chitin for the tubes (Ivanov, 1963; Southward & Southward, 1966). Within the protein cells grains are found over endoplasmic reticulum (ER in Plate 2), Golgi areas and on the secretion granules. A detailed description of the fine structure of various epidermal cells in Pogonophora is to be given elsewhere (Gupta & Little, 1969b),
These results from autoradiography provide the following conclusions relevant to the present study: (1) they clearly establish the absence of any non-specific binding or adsorption either on the surface material or in the cuticle. It will be reasonable to exclude the possibility that such a phenomenon contributes to any significant degree to the values for uptake of amino acids given above; (2) they show that a large portion of the bound 3H-phenylalanine is being incorporated into secretory materials. This material is discharged into the external medium, and is lost from the animal. It is therefore presumably contributory to the formation of a plateau or decline in the uptake curve (Text-fig. 2), since under these conditions the amino acid taken up is balanced by loss via diffusion, metabolism and secretion.
DISCUSSION
Two main points of interest arise from the results reported above. First comes the problem of the mechanism and site of uptake of organic molecules. Second is the question of how much such organic molecules might contribute to the metabolism of Pogonophora. These two points are discussed in turn.
The site and mechanism of uptake of organic molecules
Most interest in pogonophoran feeding mechanisms has so far centred upon the tentacles. Ivanov’s original suggestion (Ivanov, 1955) was that the tentacles act as a filtering mechanism, and that external digestion of the plankton caught by the tentacles precedes absorption. Following the theory of Jägersten (1957) that dissolved organic substances might be taken up, electron-microscopic studies of the tentacles (Nørrevang 1965; Gupta et al. 1966) showed an arrangement with many microvilli protruding through a cuticle, and it was suggested that nutrients could be absorbed through these microvilli. Micropinocytosis at the base of the microvilli was also observed. Southward & Southward (1966) have suggested that the subcuticular esterase found in the tentacle and anterior region of the body might be part of a system for uptake of nutrients.
The electron microscope studies show no particular specialization of the tentacular epidermis (Gupta & Little, 1969a, b) ; and since the total flux of phenylalanine through the tube walls appears to be similar to the rate of uptake of phenylalanine, it seems likely that uptake occurs along the whole length of the animal. In support of this suggestion, it may be noted that the cuticle on parts of the pyriform glands, and on the trunk region near the annulus is as thin as that on the pinnules (Gupta & Little, 1969b).
The mechanism of absorption of proteins appears to differ from that for amino acids. It involves a slow start, and this could indicate the involvement of micropinocytosis, or some predigestion by proteolytic enzymes prior to absorption. In favour of the former suggestion is the demonstration of incorporation of ferritin into the tissues by Little & Gupta (1968). The mechanism of absorption of amino acids appears to involve an uptake system similar to that shown for other marine invertebrates (e.g. Stephens, 1963), and may possibly be comparable to the systems found in some parasites (e.g. Harris & Read, 1968), where specific uptake mechanisms are found for several types of amino acid.
The general system of uptake appears to begin with diffusion through the walls of the tube, which acts as a protective filter excluding large molecules and particulate matter. Small molecules are then rapidly absorbed along the length of the body. On this basis, the tentacular apparatus favoured by Ivanov as a feeding adaptation would be mainly a respiratory adaptation.
Uptake of organic molecules and the rate of metabolism
From the graph showing uptake of phenylalanine by S. ekmani (Text-fig. 2), rates of uptake for the concentration used (2 × 10−7M, or 36 μg./l.) can be calculated. These are 0·00137 μg./mg./hr. for animals out of their tubes, and 0·00023 μg./mg./hr. for animals inside their tubes. The rate of uptake of oxygen is 0·060 μI./mg./hr. (average of figures in Table 1), and since the oxidation of 1 μg. of amino acid requires approx. 1μ. of oxygen, the phenylalanine taken up could account for 2·3 or 0·3% of the oxygen taken up, respectively for animals out of and inside their tubes. A result of a similar order seems likely for glycine, but not enough data are available for calculation.
The above figures are based on the assumption that amino acids might be present in the normal environment of Pogonophora at a concentration of about 36 μg./l. This is an average value reported for open ocean water (Degens, Reuter & Shaw, 1964; Chau & Riley, 1966). However, in the surface layers of the sediment, the concentration of organic substances due to the presence of bacterial decay is likely to be much higher. For example, Stephens (1963) found a total concentration of amino acids of about 7 × 10−6M (8000 μg./l.). If it is assumed that the ability of Pogonophora to concentrate amino acids shown in Text-fig. 2 is retained at these higher concentrations (and it is retained at 2 × 10−6 M; see Table 2), the calculations of rate of uptake can be repeated for this external concentration. They suggest that uptake of phenylalanine by animals inside their tubes could account for 70 % of the oxygen uptake, and that by animals without tubes could account for 500%. While it must be emphasized that these calculations make the assumptions that the animals are totally aerobic in their metabolism, and that they oxidize amino acids efficiently, they do suggest that the uptake of amino acids could represent an important fraction of the total metabolism.
These results can be compared with those obtained by Stephens (1963) on the polychaete Clymenella; by Ferguson (1967) on the echinoderms Asterias and Henricia-, and by Chapman & Taylor (1968) and Taylor (1969) on the polychaete Nereis. These animals all have conventional digestive systems, so that any epidermal uptake of nutrients must be considered to be supplementary to uptake through the gut. In Henricia and Asterias Ferguson suggests that the substances taken up via the epidermis may in fact only be responsible for the nutrition of epidermal tissues, since they do not appear to be passed on to the internal regions of the body. The studies of Stephens (1963) have suggested that uptake of amino acids could account for 150% of the oxygen consumption of Clymenella, but Stephens is careful to point out the various assumptions that are being made; and in a later paper (Stephens, 1968), he reduces the estimated figure to 5–1·0 % of the oxygen consumed, mainly in the light of the published values for concentrations of amino acids in sea water. Chapman & Taylor (1968) and Taylor (1969) calculate that for Nereis uptake of glutamic acid could account for 15-16% of the oxygen uptake at an external concentration of 2 × 10−5M (942·6μg./l.).
These conclusions that amino acids provide an energy source for some marine invertebrates have been challenged by Johannes, Coward & Webb (1969). From studies on the marine turbellarian, Bdelloura, they suggest that the net loss of amino acids is in fact greater than the net gain. While their argument that loss of 14C-labelled amino acids may not represent accurately the loss of total amino acids may be true, it is unfortunate that their own measurements of loss rates were made only 24 hr. after feeding. Jennings (1957) has shown that digestion and/or absorption may not be complete until 24 hr. after feeding, after which the undigested material is thrown out. Presumably the rate of loss of excretory material, including amino acids, will decline after this time. Certainly it seems unwise to assume that the experiments of Johannes et al. (1969) have shown that the net loss of amino acids is in fact greater than the net gain except when the recent products of digestion are being voided.
The utilization of other organic materials by Pogonophora seems probable. Protein can be taken up, but since this does not pass through the walls of the tube, and since it is not known whether the animals ever extend out of their tubes, the importance of this cannot be assessed. Possibly some of the smaller peptides might pass through the tube wall. The uptake of glucose seems likely, as it is concentrated by starfish (Ferguson, 1967) and by corals (Stephens, 1962). The possibility that fatty acids might be taken up has not been investigated. Parasites such as the tapeworm Hymenolepis take up fatty acids (Arme & Read, 1968), but Bailey & Fairbairn (1968) have shown that large quantities are only taken up when the fatty acids are present in bile-salt micelles. The uptake of fatty acids does not seem to have been investigated for any marine invertebrate, but the suggestion of Southward & Southward (1966) that lipid may be important in the metabolism of Pogonophora points the way for further work.
ACKNOWLEDGEMENTS
We deeply appreciate the kindness of Professor H. Brattstrom in allowing one of us (C.L.) to work at the Biological Station, Bergen. We would also like to thank Dr T. Brattegard for sending to England some specimens of S. ekmani and their tubes. We are grateful to the Biochemical Institute in Bergen and to the Department of Biochemistry in Bristol for the use of their 14C-counters. Mr G. R. Ruston has helped with the calculations of fluxes of phenylalanine. Part of the expenses of materials was defrayed by a grant from the National Institutes of Health (GM 14543-01).
References
EXPLANATION OF PLATES
Plate 1
Electron microscope autoradiographs of a section through the protosome of S. mergophorum after 20 hr. in 3H-phenylalanine. A, shows that numerous silver grains are formed over all the main tissues except the cuticle and the surface material, × 7000. B, a higher magnification view of a small portion of the cuticle with surface fuzz to show the virtual absence of autoradiographic grains even though the underlying epidermal cells have a high concentration, × 15,000. Emulsion used was Ilford L4.
Plate 2
An electron microscope autoradiograph showing a small portion of a transverse section through a metameric papilla of S. mergophorum after 20 hr. in 3H-phenylalanine. The micrograph reveals small areas of chitin-secreting cells in a pyriform gland, of muscle, and of protein secreting cells in the epidermal layer. Note that the fibrous cuticle over this part of the body is exceedingly thin, × 15,000. Emulsion used was Agfa-Gevaert NUC 307.