STUDIES ON POGONOPHORAStudies on pogonophora. Ill 761 Uptake of protein Denatured 14C-labelled Chlorella 100/JC. wa proteins disperse, , d in 200 ml. 'Millipore '-filtered sea water,

J. Exp. Biol. (1969), 51, 759-773 7 5 9With 2 plates and 2 text-figitres

printed in Great Britain

STUDIES ON POGONOPHORA

III. UPTAKE OF NUTRIENTS

BY COLIN LITTLEf AND BRIJ L. GUPTA

Biological Station, Blomsterdalen, University of Bergen, Norway,Department of Zoology, University of Bristol andDepartment of Zoology, University of Cambridge

{Received 9 May 1969)

INTRODUCTION

The phylum Pogonophora consists of marine tube-dwelling worms living mainly inareas of soft mud, at temperatures usually below io° C, and therefore often at con-siderable depths. One of the most interesting features of the phylum is the lack of anyconventional gut or digestive system (Ivanov, 1963). Ivanov has suggested that thePogonophora are filter feeders, utilizing their anteriorly placed tentacles and somemechanism of external digestion; but the scarcity of cilia in some forms, and the ap-parent lack of any cells which might produce digestive enzymes (Gupta, Little &Philip, 1966), as well as an apparent lack of proteolytic enzymes demonstrable byhistochemical methods (Southward & Southward, 1966) all cast doubt on this hypo-thesis. Jagersten (1957) revived the theory of Putter (1909) that dissolved organicsubstances might be taken up directly, and preliminary experiments by Little &Gupta (1968), and investigations by Southward & Southward (1968) have providedevidence that amino acids and even proteins can be taken into the cells. In fact the up-take of dissolved organic substances by various marine invertebrates has been demon-strated by a number of workers, although in all these cases there has been doubt as tothe 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 byStephens (1962); and for echinoderms by Stephens & Virkar (1966) and Ferguson(1964, 1967).

The present paper provides information concerning the uptake of amino acids andproteins from sea water by certain Pogonophora.

MATERIAL AND METHODS

Pogonophora were collected in the Raunefjord and the Korsfjord, near the Bio-logical Station of the University of Bergen, Espegrend, Blomsterdalen, Norway. AnAgassiz trawl was used at depths of 240 m. in the Raunefjord and 680 m. in theKorsfjord. The bottom temperature is 6-70 C. Pogonophora caught in the chains andin the net were quickly transferred to sea water at 50 C, and returned to the laboratory

t Present address: Department of Zoology, University of Bristol, Bristol 8.

760 COLIN LITTLE AND BRIT L. GUPTA

of the Biological Station, where they were kept in 2 1. containers at the same temperalture. Survival was excellent for periods of 2-3 weeks, but all experiments were carriedout 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 thephysiological work was Siboglinum ekmam Jagersten, but some measurements of rateof respiration were made with S. fiordicum.

The material for autoradiography was obtained from Miami, U.S.A. Siboglinummergophorum were dredged from 200 m. off Miami Beach, Florida, where the bottomtemperature is about 8° C. Animals were removed from the dredge, placed in seawater at 8° C, transported back to the Institute of Marine Sciences, University ofMiami, 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 withtubes cleaned, rinsed in 'Millipore'-filtered sea water and with excess tube trimmedaway, were placed in ground-glass stoppered bottles of 40-70 ml. capacity also con-taining ' Millipore '-filtered sea water (pore size 0-45 fi). The bottles were placed in thedark at 5° C, and the oxygen content of the sea water was measured at the start of theexperiments and after 52-110 hr., using the Alsterberg (azide) modification of theWinkler method for dissolved oxygen. Readings were also taken for simultaneousblanks.

At the end of each experiment the worms were removed from their tubes using fineforceps 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 establishedby extrapolation back to zero time. This measurement of wet weight is liable to in-corporate a large degree of inaccuracy due to the unknown quantity of water remainingattached 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 errorsin 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 theirtubes or after these had been removed, into 1-10 ml. of solutions of 14C-phenylalanineor 14C-glycine (Radiochemical Centre, Amersham) in ' Millipore'-filtered sea water.They were kept in the dark at 50 C for periods of 5-60 min. After this time they wereremoved 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 /i\. of 80% ethyl alcohol. 50 or75 /il. of this extract was then transferred to a planchet, as were samples of the seawater 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, and14C was counted using a Frieseke and Hoepfner gas-flow counter. In general the timefor 1000 counts was taken 2-3 times, but for active samples 10,000 counts were taken.Background was approximately 14 counts/min.

Studies on pogonophora. Ill 761

Uptake of protein

Denatured 14C-labelled Chlorella protein, 100/JC., was dispersed in 200 ml.'Millipore '-filtered sea water, to which had been added 2 mg. % penicillin G and2 mg. % streptomycin sulphate to prevent bacterial and fungal action. The degree ofuniformity of dispersion of the protein can be judged from the s.E. of samples takenbefore and after experiments: 1570 counts/min./io/tl. + 123*5 (20 samples). Pogono-phora were placed individually in 10 ml. of this protein suspension for periods of 10 or60 min., and were then rinsed as above, and macerated on planchets. After dryingdown they were counted for 14C directly.

In all these experiments, corrections have been made for self-absorption and forbackground.

Permeability of the tubes to phenylalanine

Empty tubes were placed in a damp chamber, and 2 x IO~*M 14C-phenylalanine in' Millipore '-filtered sea water was injected from a microburette. The ends were sealedwith dental wax, and the tubes were immersed in small glass tubes containing 500 fi\.of sea water, at 50 C. This was stirred by blowing air in through a fine glass tube.Samples of sea water, 10 /A., were taken at intervals (from 1 min. to 6 hr. after im-mersion), dried down on planchets, and counted for 14C on a Nuclear-Chicago gas-flowcounter. The figures obtained were adjusted for background, self-absorption and forvolume changes incurred by removal of samples.

The dimensions of the tubes, which were approximately 5 cm. long and o-oi cm. indiameter, with a volume of 0-5-1-0 /A., were measured using a binocular microscope(magnification X200).

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 concentrationinside the tube was therefore 1 x I O ^ M . The flux,/, was calculated from this slope.The permeability coefficient, P, was then calculated from

/ = PAC,

where A is the area of the tube in cm.2, estimated using the average of internal andexternal radii, and C is the concentration in M/cm.3. The assumption was made thatP would be similar at lower concentrations than IO~*M, and the flux at 2 x IO~7M wascalculated.

Autoradiography

Specimens of Siboglinum mergophorum from Miami, Florida, still enclosed in theirtubes, were placed in 2 XIO~6M 8H-phenylalanine (s.A. 5-0 Ci/mM) in ' Millipore'-filtered sea water. The final concentration of the label was 100/iCi/ml. sea water(35 %o S) at 8° C. Specimens were removed either after 1 hr. or after 20 hr. To processthe material for microscopy, animals were removed from their tubes, rinsed in severalchanges of filtered sea water, and fixed for 2 hr. in 4 % glutaraldehyde (Biological grade)in o-1 M sodium cacodylate buffer at pH 7-4 with added sucrose to give a final measuredconcentration of 1100 m-osmoles/1. Fixed animals were then cut into suitably smallpieces, washed thoroughly in several changes of sucrose containing cacodylate buffer

762 COLIN LITTLE AND BRIJ L. GUPTA

(IIOO m-osmoles/1., overnight at 8° C), post-fixed in buffered OsO4, dehydrated andembedded in ' Araldite' (Gupta et al. 1966; Gupta & Little, 1969a).

For light-microscopic autoradiography, 0-5 fi thick sections of the 'Araldite' blocksfrom various regions of the animals were mounted on gelatine-subbed glass slides andcoated with Ilford L4 liquid nuclear track emulsion. The details of such a procedureare given by Rogers (1967). All the requisite controls for chemography and otherartifacts (Rogers, 1967) were used. Autoradiograms were exposed from 2 days to10 weeks, developed in Kodak Di9b or D170 for 3-5 min. at 200 C, fixed in 25%sodium thiosulphate, washed and dried. They were examined either unstained byphase-contrast and dark-ground microscopy, or stained briefly in 0-5 % toluidine bluethrough the gelatine.

For electron microscopy, 500-1000 A thick sections were mounted on glass slidescoated 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 eitherIlford L4 (purple interference colour) or Agfa-Gevaert NUC 307 (pale gold inter-ference colour) nuclear track emulsions, using a Kopriwa-type semi-automatic coatingmachine (Kopriwa, 1967a). Under our laboratory conditions, the dilution needed was2:3 for Ilford L4 and 3:2 for Gevaert NUC 307; both emulsions being used at 350 Cand 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 40 Cfor 6-12 weeks. Autoradiograms were processed in an appropriate developer for eachemulsion (Kopriwa, 1967 ft), fixed and washed. The section-bearing areas of the celloi-din films were floated on water and mounted on naked copper grids for electron micro-scopy (Salpeter & Bachmann, 1964; Rogers, 1967). When thoroughly dry, grids wereexamined, without any further treatment, in a Philips EM 200 electron microscopefitted 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 thedirect uptake of organic substances in the metabolism of Pogonophora, crude measure-ments were made of the rate of uptake of oxygen, using groups of 10-14 animals foreach determination. Animals were used in their tubes, and these often have protozoaattached, or harbour bacteria and fungi. In two series of experiments animals wereplaced for 2 hr. in either 2 mg. % penicillin G and 2 mg. % streptomycin sulphate, orin o-oi mg. % chloromycetin, before transference to the closed bottles for measure-ment of oxygen uptake. Results are shown in Table 1, and suggest that neither bacterianor fungi on the tubes contribute significantly to the rate of uptake of oxygen by thePogonophora.

These results are of the same order as those found by Manwell, Southward &Southward (1966) for the larger form SibogKnum atlanticum. For example, a singledetermination made by these workers soon after collection of the specimen gave aresult of 0-1203 ml. Og/hr./gm. wet weight, at a temperature of 150 C, as compared toa value of 0060 ml. Oj/hr./gm. for S. ekmani at 50 C.

Studies on pogonophora. Ill 763

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 ahigher specific activity than glycine. Initially, the relationship between weight and rateof uptake was briefly investigated. With an experiment time of 10 min., and using2 x IO~7M phenylalanine, the amount of radioactivity in 75 fil. of alcohol extract wasplotted against the wet weight of animals extracted (Text-fig. 1). Since the scatter was

Table 1. Oxygen consumption of Siboglinum ekmani andS. fiordicum at 50 C

Species

S. fiordicumS. ekmaniS. ekmaniS. ekmani

Av. wt. mg.

1-48064058O-53

100r

80

£ 602

Rate of uptake of O,/d./mg./hr. ± s.E.

(no. of observations)

o°33 — (2)0-052 ±0-0054 (6)0-067±00104 (4)0061 ±0-0101 (4)

Antibiotics added

Penicillin + streptomycinChloromycetin

40

E6.

20

0-2 0-4 0-6Wt.-mg.

08 10

Text-fig. 1. The relation between quantity of "C-phenylalanine taken upand weight, in S. ekmani.

very great, and since most of the animals used in subsequent experiments were be-tween 0-3 and o-6 mg., adjustment for variation in weight was made by dividing theradioactivity of the extract by the weight of the animal. This figure was then expressedas counts/min./mg. (wet wt.) of animal, and was compared directly with the figure forcounts/min.//il. sea water, to give a 'concentration factor'. The true relationship ofweight to amino acid taken up is probably logarithmic but in the present case there istoo much scatter to decide this, and the linear relation in fact approximates to thelogarithmic one over the range of weights used.

The comparison between animals in their tubes and those taken out may be criti-cized on the basis that damage may occur during the extraction from the tube; althoughno visible damage occurred. Similarly, all the experiments may be criticized because fewof the animals were complete; although those that lack the postannular region appear

764 COLIN LITTLE AND BRIJ L. GUPTA

to seal off the break behind the annulus. Against such criticism, it may be pointed ouHthat since the animals actually concentrate amino acids in a relatively short time, theimportance of diffusion through wounds and damaged areas, which would only occurdown concentration gradients, must be negligible.

(a) Phenylalanine

A concentration of 2 x IO~7M was employed, as this represents 36 /tg./l. (at the S.A.used), this value being of the same order as the observed total concentration of aminoacids in sea water (Degens, Reuter & Shaw, 1964; Chau & Riley, 1966). Results ob-tained over a time of 60 min. are shown in Text-fig. 2 for S. ekmam, both when in their

x20

X15

x10

X5

20 40Time (mln.)

60

Text-fig. 2. The relation between quantity of "C-phenylalanine taken up, expressed as a con-centration factor, and time, in S. ekmam. • , Animals in their tubes. O, Animals removedfrom their tubes. Vertical lines represent twice the standard error of the mean. Diagonal linesrepresent the initial rate of uptake.

tubes and when removed from their tubes. It is evident that with the animals removedfrom their tubes a high uptake rate occurs up to 20-30 min., and that this falls off afterlonger periods. The fall-off is presumably due to a balance being reached betweenuptake and loss via diffusion, metabolic breakdown and secretion. The rate of uptakeby animals still in their tubes is much slower, and over 1 hr. the curve appears to belinear; the preliminary results of Little & Gupta (1968) show that concentrationscomparable to those produced by animals without tubes can be reached over periodsof 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 x IO~7M phenylalanine with2 mg. % penicillin G and 2 mg. % streptomycin sulphate to stop bacterial action.Second, the concentration of phenylalanine was increased to 2 x 1 o"6 M, and observationswere made of uptake after 10 min. These values are compared with those for 10 min.experiments at 2 x io~7 M with no added antibiotics, in Table 2. There appears to be

Studies on pogonophora. HI 765

po effect on uptake when antibiotics are added, suggesting that bacteria are not in-volved in the uptake process. With a tenfold increase in concentration, concentrationfactors are similar to those at 2 x IO~7M.

Measurements of the amount of radioactivity incorporated into the alcohol-insoluble fraction during all these experiments showed that only 3-4% of the 14Ctaken up was incorporated into this fraction. There appeared to be no significantpercentage increase in the alcohol-insoluble fraction with increased time of exposureto amino acids. The figures for S. ekmani out of their tubes are given in Table 3, andare of the same order as those given by Stephens & Virkar (1966) for the brittle starOpMactis.

Table 2. Uptake of phenylalanine by S. ekmani removed fromtheir tubes, in a 10 min. period

Cone, ofphenylalanine

2 X IO~TM2 X IO~7M

a x IO~*M

Av. wt. of Cone, factor + 8.E.Antibiotics added

2 mg. % streptomycin2 mg. % penicillin

animals mg. ± s.E, (no. of observations)

0-48 ±0-0700-35 ±°-O43

0-48 ±0-090

7-2o± 1-089 (11)9-i8±o-84o(5)

6-44 ±2-544 (5)

Table 3. Percentage qf14C taken up into the alcohol-insoluble fraction byS. ekmani without their tubes

Significance of difference

Amino acid

Phenylalanine

Glycine

Exposuretime

(min.)

10601060

Alcohol-insol. 14C as% total ltC taken up

+ s.E. (no. of observations)

3-69±0974 (11)4- IO±I -6O8 (5)

5-94 ±i-425 (5)4-79 ±i-575 (5)

between 10 and 60 min. value

t

02278

0-5160

P

> o-io

> o-io

Time-min.

10601060

Table 4. Uptake of glycine by S. ekmani

Presence (I)or absence (O)

Cone, of glycine of tubes Av. wt. mg. ± s.E.

6x IO~7M6x IO~7M6x IO~'M6x IO~'M

OOII

0-52 ± 0-0540-67 ±0-0900-52 ±0087O-59±O-I22

Cone, factor ±s.B.(no. of observations)

478±0097 (5)22-42 ±5264 (5)208±0562 (5)640±0677 (5)

(b) GlycineA concentration of 6 x IO~7M (or 46-8 /ig./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 obtainedwith phenylalanine, except that the uptake after 10 min. by animals inside their tubesis higher for glycine than for phenylalanine: for phenylalanine, the concentrationfactor is 1-15 + 0-477 (5). ̂ d f o r glycine it is 2-98 ± 0-562 (5) (t = 2-453, P = 0-05 —0-02).

766 COLIN LITTLE AND BRIJ L. GUPTA

Measurements of the alcohol-insoluble fraction were similar to those obtained withphenylalanine (Table 3), and suggest that very little of the accumulated carbon is in-corporated 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 x io~7 M are given in Table 5. Thelength 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 cantherefore be directly compared with the figures for uptake of phenylalanine by animalswithin their tubes. The slope of the relevant line in Text-fig. 2 corresponds to an up-take rate of 0-00023 Pg-lmg- animal/hr. Although the figures for flux rates are very

Table 5. Permeability of the tubes of S. ekmani to phenylalanine

Anterior (A) or Permeabilityposterior (P) coefficient P, Flux at 2 x IO~'M

Tube region (Px 10* cm. sec."1) /tg./tube/hr.

2

67845910

AAAAPPPP

1958-96-5Si

6-624-68-6

47-0

0-0006310-0002330-0001510-000120

0 000124

0-0007430-0001760000880

variable, they are of the same order as this, suggesting that the rate of passage throughthe wall of the tube is the main limiting factor controlling uptake; and that the passageof amino acid in from the end of the tube is not greatly significant. This hypothesisgains support from some brief experiments using dyes. Small amounts of methyleneblue were injected into the lumen of tubes containing living S. ekmani; but althoughthe animals moved slowly up and down their tubes the dye had not moved appreciablyafter 12 hr., showing that no ciliary current moves into or out of the aperture of thetube.

When the figures for protein are considered (see next section), it is obvious that noprotein is penetrating the tube walls. The tube is, in fact, acting as a filter, allowingthrough only small molecules. This is a strikingly similar phenomenon to that shownby the rectal intima of the locust ScMstocerca (Philips & Dockrill, 1968), which allowsthe passage of small molecules, but prevents the passage of inulin and proteins. Thecomparison is even more interesting when it is noted that both membranes are chiti-nous, and that the permeability coefficients of the rectal intima for small molecules aresimilar 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 5. ekmani to take up protein, individual animals wereplaced in a suspension of denatured Chlorella protein (Radiochemical Centre, Amer-sham; 'freed of water-soluble compounds by molecular filtration'), to which wasadded 2 mg. % penicillin G and 2 rag. % streptomycin sulphate to prevent bacterial

Studies on pogonophora. Ill 767

breakdown. The concentration used was 500/tc/l., or iooo/ig./L, ± 5%. The resultshave been calculated in a similar way to those for amino acids, but although the proteinis in suspension, the dispersion is not perfect (see Methods).

Table 6 shows the results, from which it is obvious that the situation is somewhatdifferent from that obtained with amino acids. In 10 min. animals out of their tubestake 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 takessome 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 acidsolution. 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 itis not drawn in through the openings at the ends of the tube.

Table 6. Uptake of protein by S. ekmani

(500 /JC./I., approx. 1000 /*g./l.)

Av. wt of Presence (I) or Cone factor ± s.E.Time-min. animnla rag. ± s.E. absence (O) of tubes (no. of observations)

10 o-50±o-o89 O o-i4±o-09(5)60 o-40±o-oso O ii-S9±a-Si Is)60 0-55 ±0-092 I 0-03 ±o-oi (5)

Autoradiography

The technique of autoradiography employed here will demonstrate only that por-tion of the total sH-phenylalanine taken up by the animal which is either bound or hasbeen incorporated into molecules that survive subsequent processing of the tissue. Thisfraction will probably be similar to the alcohol-insoluble fraction in the data givenabove, and will therefore probably not constitute more than 5 % of the total uptake, atleast over the short time range of 1 hr. The technique, therefore, does not indicate thesites of uptake but the sites of metabolic incorporation into the tissue and cell com-ponents. In the final autoradiographs such sites of incorporation are indicated by thepresence 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 bebetter than 2000 A with Ilford L4 emulsion, and better than 1000 A with Agfa-Gevaert NUC 307 emulsion (Bachmann, Salpeter & Salpeter, 1968).

Light-microscope autoradiographs provide information on gross localization of thelabel over different parts of the animal, and preliminary results from such preparationshave already been published (Little & Gupta, 1968; Southward & Southward, 1968).In such preparations, silver grains are formed over the tentacle as well as all over themain body of the animal as far back as the annulus. No material was available from theregion posterior to the annulus. High concentrations of grains occur over areas wherehigh metabolic or synthetic activity is expected, such as the base of the tentacles andthe cephalic lobe, in the forepart (Little & Gupta, 1968), and in the region of the meta-meric papillae and the ovary in the trunk. Even at high magnifications the surface ofthe animals, the cuticle, and the ciliary band do not show any significant binding ofthe label; the grain density over these regions being no higher than the backgroundlevel.

768 COLIN LITTLE AND BRIJ L. GUPTA

High-resolution autoradiographs examined by electron microscopy confirm

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

Table 7. Relative distribution of autoradiographic grains over different tissues of

S. mergophorum kept for 1 hr. in 2 XIO~*M zH-phenylalanine

Tissue

BackgroundTotal tissue scannedCuticleBloodNerveMuscleEpidermal cells*

Autoradio-graphicgrains/100/*•

2-50

41-003 4°

16-0017-0025 0074-00

Total area(tissue)scanned

(%)

2O-OO

IOO

I2-5O

15-25762

20-504420

Totalgrains over

tissue(%)

I-2O

IOO

I'2I6-oi3-05

123177-42

Grains (%)/area (%)

0-06

i-oo0-09O390-400 6 0

i-8o

Emulsion used: Ilford L4• These cells were mostly ordinary epidermal cells from the tentacles and the protosome but some

mucous cells may have been included. The density of autoradiographic grains over pinnule cells was thesame and these cells have also been counted as ordinary epidermal cells in the present data.

Table 8. Relative distribution of autoradiographic grains over different tissues ofS. mergophorum kept for 20 hr. in 2 x IO~6M 3H-pkenylalanine

Tissue

BackgroundTotal tissue scannedCuticleBloodMuscleNerveOrdinary epidermal cellsCilary cell bodies*Mucous cellsChitin cellsfProtein cells

Autoradio-graphicgrains/IOO/i1

2 0

19324

1 2 0

2 7 0

1772 1 4

107

225

79733

Totalarea scanned

(%)

2O-OO

ioo-co7-207-50

23-50636

25-0015-70

i-8o9-603 4O

Totalgrains(%)

2-2O

ioo-ooO-924-80

32605 9 0

26-OO

1O-7O

2-:o4 0 0

13-00

Grains (%)/area (%)

O-II

i-oo0 1 30-641-400-921-04o-681 1 7

0 4 1

4 0 0

Emulsion used: Agfa-Gevaert NUC 307.• These cells form a ciliary band in the metasome. The density of autoradiographic grains over the

cilia and the cuticle through which they protrude was comparable to that given above for the rest of thecuticle.

t These cells form the pyriform glands which are presumed to secrete chitin (Southward & South-ward, 1966). In the autoradiographs of these cells most of the area was occupied by the secretion in thelumen of the glands.

these cells). Tables 7 and 8 summarize the distribution patterns of autoradiographicgrains 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 /i2 of thetissue in sections and as a ratio of the percentages of total grains counted and of totalarea occupied by a particular tissue in all the electron autoradiographs examined in the

Studies on pogonophora. HI 769

present study. This last value permits a direct comparison of the relative incorporationBf 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 bindingof the label in the cuticle and associated surface material even after a 20 hr. exposure to3H-phenylalanine (PI. 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 themaximum concentration occurs in the epidermal cells which in this case include thepinnule cells of the tentacles. A separate analysis of the grain distribution over thepinnules did not reveal any significant difference in grain/area ratio from the rest of theepidermal cells.

In specimens kept in the label for 20 hr. the general pattern of grain distribution ismaintained (Table 8). Furthermore, different cell types in the epidermis show a widerange of radioactive incorporation. A maximum concentration of grains is found overthe cells which by their distribution in the animal are identified as ' protein secretingcells' in the metameric papillae of the metasome (Plate 2). These cells probably cor-respond to the 'white protein cells' described in S. atlanticum and histochemicallyanalysed by Southward & Southward (1966) as producing a tyrosine-rich proteinsecretion. The lowest grain/area ratio is found over the secretion contained in thelumina 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 overendoplasmic reticulum (ER in Plate 2), Golgi areas and on the secretion granules. Adetailed description of the fine structure of various epidermal cells in Pogonophora isto be given elsewhere (Gupta & Little, 19696),

These results from autoradiography provide the following conclusions relevant tothe present study: (1) they clearly establish the absence of any non-specific binding oradsorption either on the surface material or in the cuticle. It will be reasonable to ex-clude the possibility that such a phenomenon contributes to any significant degree tothe values for uptake of amino acids given above; (2) they show that a large portion ofthe bound 3H-phenylalanine is being incorporated into secretory materials. Thismaterial is discharged into the external medium, and is lost from the animal. It istherefore presumably contributory to the formation of a plateau or decline in the up-take curve (Text-fig. 2), since under these conditions the amino acid taken up isbalanced by loss via diffusion, metabolism and secretion.

DISCUSSION

Two main points of interest arise from the results reported above. First comes theproblem of the mechanism and site of uptake of organic molecules. Second is thequestion of how much such organic molecules might contribute to the metabolism ofPogonophora. 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 thetentacles. Ivanov's original suggestion (Ivanov, 1955) was that the tentacles act as afiltering mechanism, and that external digestion of the plankton caught by the tentaclesprecedes absorption. Following the theory of Jagersten (1957) that dissolved organic

770 COLIN LITTLE AND BRIJ L. GUPTA

substances might be taken up, electron-microscopic studies of the tentacles (Narrevanaj1965; Gupta et al. 1966) showed an arrangement with many microvilli protrudingthrough a cuticle, and it was suggested that nutrients could be absorbed through thesemicrovilli. Micropinocytosis at the base of the microvilli was also observed. Southward& Southward (1966) have suggested that the subcuticular esterase found in the tentacleand 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 tentacularepidermis (Gupta & Little, 19690,6); and since the total flux of phenylalanine throughthe tube walls appears to be similar to the rate of uptake of phenylalanine, it seemslikely that uptake occurs along the whole length of the animal. In support of thissuggestion, it may be noted that the cuticle on parts of the pyriform glands, and on thetrunk region near the annulus is as thin as that on the pinnules (Gupta & Little, 1969^).

The mechanism of absorption of proteins appears to differ from that for aminoacids. It involves a slow start, and this could indicate the involvement of micropino-cytosis, or some predigestion by proteolytic enzymes prior to absorption. In favour ofthe former suggestion is the demonstration of incorporation of ferritin into the tissuesby Little & Gupta (1968). The mechanism of absorption of amino acids appears toinvolve 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 para-sites (e.g. Harris & Read, 1968), where specific uptake mechanisms are found forseveral types of amino acid.

The general system of uptake appears to begin with diffusion through the walls ofthe tube, which acts as a protective filter excluding large molecules and particulatematter. Small molecules are then rapidly absorbed along the length of the body. Onthis basis, the tentacular apparatus favoured by Ivanov as a feeding adaptation wouldbe mainly a respiratory adaptation.

Uptake of organic molecules and the rate of metabolism

From the graph showing uptake of phenylalanine by S. ekmcan (Text-fig. 2), rates ofuptake for the concentration used (2 x IO~7M, or 36 /tg./l.) can be calculated. These are0-00137 /ig7mg-/nr- f°r animals out of their tubes, and 0-00023 /ig./mg./hr. for animalsinside their tubes. The rate of uptake of oxygen is 0-060 /il./mg./hr. (average of figuresin Table 1), and since the oxidation of 1 fig. of amino acid requires approx. 1 /A. ofoxygen, the phenylalanine taken up could account for 2-3 or 0-3 % of the oxygentaken up, respectively for animals out of and inside their tubes. A result of a similarorder 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 inthe normal environment of Pogonophora at a concentration of about 36 fig./l. This isan average value reported for open ocean water (Degens, Reuter & Shaw, 1964; Chau6 Riley, 1966). However, in the surface layers of the sediment, the concentration oforganic 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 about7 x IO~6M (8000 fig./I.). If it is assumed that the ability of Pogonophora to concentrateamino acids shown in Text-fig. 2 is retained at these higher concentrations (and it isretained at 2 x icr6 M; see Table 2), the calculations of rate of uptake can be repeatedfor this external concentration. They suggest that uptake of phenylalanine by animals

Studies on pogonophora. Ill 771

fcside their tubes could account for 70 % of the oxygen uptake, and that by animalswithout tubes could account for 500%. While it must be emphasized that these cal-culations 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 aminoacids could represent an important fraction of the total metabolism.

These results can be compared with those obtained by Stephens (1963) on thepolychaete Clymenella; by Ferguson (1967) on the echinoderms Asterias and Henricia;and by Chapman & Taylor (1968) and Taylor (1969) on the polychaete Nereis. Theseanimals all have conventional digestive systems, so that any epidermal uptake ofnutrients must be considered to be supplementary to uptake through the gut. InHenricia and Asterias Ferguson suggests that the substances taken up via the epidermismay in fact only be responsible for the nutrition of epidermal tissues, since they do notappear 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 theoxygen consumption of Clymenella; but Stephens is careful to point out the variousassumptions that are being made; and in a later paper (Stephens, 1968), he reduces theestimated figure to 5-1 o % of the oxygen consumed, mainly in the light of the publishedvalues for concentrations of amino acids in sea water. Chapman & Taylor (1968) andTaylor (1969) calculate that for Nereis uptake of glutamic acid could account for15—16% of the oxygen uptake at an external concentration of 2 XIO~5M (942-6 /ig./l.).

These conclusions that amino acids provide an energy source for some marine in-vertebrates have been challenged by Johannes, Coward & Webb (1969). From studieson the marine turbellarian, Bdelloura, they suggest that the net loss of amino acids is infact greater than the net gain. While their argument that loss of 14C-labelled aminoacids may not represent accurately the loss of total amino acids may be true, it isunfortunate that their own measurements of loss rates were made only 24 hr. afterfeeding. Jennings (1957) has shown that digestion and/or absorption may not be com-plete 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 declineafter this time. Certainly it seems unwise to assume that the experiments of Johanneset al. (1969) have shown that the net loss of amino acids is in fact greater than the netgain except when the recent products of digestion are being voided.

The utilization of other organic materials by Pogonophora seems probable. Proteincan be taken up, but since this does not pass through the walls of the tube, and since itis not known whether the animals ever extend out of their tubes, the importance of thiscannot be assessed. Possibly some of the smaller peptides might pass through the tubewall. 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 takenup has not been investigated. Parasites such as the tapeworm Hymenolepis take upfatty acids (Arme & Read, 1968), but Bailey & Fairbairn (1968) have shown that largequantities 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 marineinvertebrate, but the suggestion of Southward & Southward (1966) that lipid may beimportant in the metabolism of Pogonophora points the way for further work.

49 Exp. Biol. 51, 3

772 COLIN LITTLE AND BRIJ L. GUPTA

SUMMARY

1. The oxygen consumption of S. ekmani at 50 C is 0-06 /il./mg./hr.2. Phenylalanine and glycine are concentrated by 5. ekmani. The concentration

factor reaches a maximum after 30 min. in animals removed from their tubes. Inanimals inside their tubes, the rate of uptake is limited by the rate of diffusion throughthe walls of the tube. The phenylalanine does not move appreciably into the alcohol-insoluble extract of the animals over a period of 1 hr.

3. Protein is taken up by 5. ekmani when animals are removed from their tubes.Uptake is slower than uptake of amino acids, and may involve a different mechanism.

4. Autoradiography using 5. mergophorum shows that phenylalanine is not adsorbedon the cuticle. It is found especially in secretory cells, within which it is localized overrough endoplasmic reticulum, Golgi regions and secretion spherules.

5. The site and mechanism of uptake of organic molecules are discussed; and thetypes of molecules absorbed, together with the significance these may have in theoverall metabolism, are considered.

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 DrT. 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 ofBiochemistry in Bristol for the use of their 14C-counters. Mr G. R. Ruston has helpedwith the calculations of fluxes of phenylalanine. Part of the expenses of materials wasdefrayed by a grant from the National Institutes of Health (GM 14543-01).

REFERENCES

AHME, C. & READ, C. P. (1968). Studies on membrane transport: IT. The absorption of acetate andbutyrate by Hymenolepis dimtnuta (Cestoda). Biol. Bull. mar. biol. Lab., Woods Hole 135, 80-91.

BACHMANN, L., SALPETBR, M. M. & SALPETER, E. E. (1968). Das AusftSsungsvermflgen elektronen-mikroakopischer Autoradiographen. ffiitochemie 15, 234—50.

BAILEY, H. H. & FAIRBAIRN, D. (1968). Lipid metabolism in helminth parasites. V. Absorption of fattyacids and monoglycerides from micelkr solution by Hymenolepis dimmita (Cestoda). Comp. Biochan.Phytiol. 36, 819-36.

CHAPMAN, G. & TAYLOR, A. G. (1968). Uptake of organic solutes by Nereis virens. Nature, Lond. 217,763-4-

CHAU, Y. K. & RILEY, J. P. (1966). The determination of amino acids in sea water. Deep Sea Res. 13,1115-24.

DECENS, E. T., REUTER, J. H. & SHAW, K. N. F. (1964). Biochemical compounds in offshore Californiasediments and seawaters. Geochim. cosmoc/u'm. Acta 28, 45-66.

FERGUSON, J. C. (1964). Nutrient traruport in Starfish. II. Uptake of nutrients by isolated organs. Biol.Bull. mar. biol. Lab., Woods Hole 126, 391-406.

FERGUSON, J. C. (1967). Utilization of dissolved exogenous nutrients by the starfishes, Asterias forbesiand Henrida sanguinolenta. Biol. Bull. mar. biol. Lab., Woods Hole 132, 161-73.

GUPTA, B. L. & LITTLE, C. (1969a). Studies on Pogonophora. II. Ultrastructure of the tentacular crownof Siphonobracfua. J. mar. biol. Ass. UJC. 49, 717-41.

GUPTA, B. L. & LITTLE, C. (19696). Studies on Pogonophora. IV Fine structure of the cuticle and epider-mis. Tissue Sf Cell. (In the Press.)

GUPTA, B. L. & LITTLE, C. (1969c). Studies on Pogonophora. V. Fine structure of the tube and associatedtissues. Tissue & Cell. (In the Press.)

GUPTA, B. L., LITTLE, C. & PHILIP, A. M. (1966). Studies on Pogonophora. Fine structure of the ten-tacles. J. mar. biol. Ass. U.K. 46, 351-72.

HARRIS, B. G. & READ, C. P. (1968). Studies on membrane transport. III. Further characterization ofamino acid systems in Hymenolepis diminuta (Cestoda). Comp. Biochem. Physiol., 26, 545—52.

Journal of Experimental Biology, Vol. 51, No. 3

Cuticle

Plate 1

B

C. LITTLE AND B. L. GUPTA (Facing p. 772)

Journal of Experimental Biology, Vol. 51, No. 3

Surface

V

Plate 2

Cuticle

*

ER

•>' ' Chitin in

lumen

C. LITTLE AND B. L. (,L"PTA

Studies on pogonophora. Ill 773V, A. V. (1955). The main features of the organisation of Pogonophora. On external digestion in

Pogonophora. On the assignment of class Pogonophora to a separate phylum of Deuterostomia—Brachiata. A. Ivanov, phyl. nov. Syst. Zool. 4, 171-7 (Translation by A. Petrunkevitch).

IVANOV, A. V. (1963). Pogonophora. Translated into English by D. B. Carlisle. London: Academic Press.JACERSTEN, G. (1957). On the larva of Siboglimtm, with some remarks on the nutrition problem of the

Pogonophora. Zool. Bidr. Uppi. 33, 67-79.JENNINGS, J. B. (1957). Studies on feeding, digestion, and food storage in free-living flatworms

(Platyhelminthes: Turbellaria). Biol. Bull. mar. biol. Lab., Woods Hole 113, 63-80.JOHANNES, R. E., COWARD, S. J. & WEBB, K. L. (1969). Are dissolved amino acids an energy source for

marine invertebrates? Comp. Biochem. Pkytiol. 39, 283-8.KOPRIWA, B. M. (1967a). A semiautomatic instrument for the radioautographic coating technique. J.

Hittochem. Cytochem. 14, 923-8.KOPRIWA, B. M. (19676). The influence of development on the number and appearance of silver grains

in electron microscopic radioautography. J. Histochem. Cytochem. 15, 501-15.LITTLE, C. & GUPTA, B. L. (1968). Pogonophora: uptake of dissolved nutrients. Nature, Land. 318,

873-4-MANWELL, C, SOUTHWARD, E. C. & SOUTHWARD, A. J. (1966). Preliminary studies on haemoglobin and

other proteins of the Pogonophora. J. mar. biol. Ass. U.K. 46, 115-24.N0RREVANG, A. (1965). Structure and function of the tentacle and pinnules of Siboglmum ekmani

JSgersten (Pogonophora). Sartia, 31, 37-47.PHILLIPS, J. E. & DOCKRILL, A. A. (1968). Molecular sieving of hydrophilic molecules by the rectal

intima of the desert locust (Schistocerca gregaria). J. exp. Biol. 48, 521-32.PUTTER, A. (1909). Die ErnaJtrung der Wassertxere und der Stoffhaushalt der Gewasser. Fischer, Jena.ROGERS, A. W. (1967). Techniques of Autoradiography. Elaevier, Amsterdam.SALPETER, M. M. & BACHMANN, L. (1964). Autoradiography with the electron microscope. A procedure

for improving resolution, sensitivity, and contrast. J. Cell. Biol. 32, 469—77.SOUTHWARD, E. C. & SOUTHWARD, A. J. (1966). A preliminary account of the general and enzyme histo-

chemistry of Siboglinum atlanticum and other Pogonophora. J. mar. biol. Ass. U.K. 46, 579-616.SOUTHWARD, A, J. & SOUTHWARD, E. C. (1968). Uptake and incorporation of labelled glycine by pogono-

phores. Nature, Lond. 318, 875-6.STEPHENS, G. C. (1962). Uptake of organic material by aquatic invertebrates. I. Uptake of glucose by the

solitary coral, Fungia scutaria. Biol. Bull. mar. biol. Lab., Woods Hole 133, 648-59.STEPHENS, G. C. (1963). Uptake of organic material by aquatic invertebrates. II. Accumulation of

amino acids by the bamboo worm, Clymenella torquata. Comp. Biochem. Physiol. 10, 191—202.STEPHENS, G. C. (1964). Uptake of organic material by aquatic invertebrates. III. Uptake of glycine by

brackish-water annelids. Biol. Bull. mar. biol. Lab., Woods Hole 136, 150-62.STEPHENS G. C. (1968). Dissolved organic matter as a potential source of nutrition for marine organisms.

Am. Zool. 8, 95-106.STEPHENS, G. C. & SCHINSKE, R. A. (1961). Uptake of amino acids by marine invertebrates. Limnol.

Oceanogr. 6, 175-81.STEPHENS, G. C. & VIRKAR, R. A. (1966). Uptake of organic material by aquatic invertebrates. IV. The

influence of salinity on the uptake of amino acids by the brittle star, Ophiactis arenosa. Biol. Bull. mar.biol. Lab., Woods Hole 131, 172-85.

TAYLOR, A. G. (1969). The direct uptake of amino acids and other small molecules from seawater byNereis virens Sars. Comp. Biochem. Physiol. 39, 243-50.

EXPLANATION OF PLATES

PLATE I

Electron microscope autoradiographs of a section through the protosome of S. mergophorum after 20 hr.in *H-phenylalanine. A, shows that numerous silver grains are formed over all the main tissues exceptthe cuticle and the surface material, x 7000. B, a higher magnification view of a small portion of thecuticle with surface fuzz to show the virtual absence of autoradiographic grains even though the under-lying epidermal cells have a high concentration, x 15,000. Emulsion used was Ilford L4.

PLATE 2

An electron microscope autoradiograph showing a small portion of a transverse section through ametameric papilla of S. mergophorum after 20 hr. in 'H-phenylalanine. The micrograph reveals small areasof chitin-secreting cells in a pyriform gland, of muscle, and of protein secreting cells in the epidermallayer. Note that the fibrous cuticle over this part of the body is exceedingly thin, x 15,000. Emulsionused was Agfa-Gevaert NUC 307.

49-2