studies on taurine in the euryhaline bivalve mya arenaria

Camp. Biochem. Physiol., 1972, Vol. 4lA, pp. 307 to 317. Pergamon Press. Printed in Great Britain

STUDIES ON TAURINE IN THE EURYHALINE BIVALVE MYA ARENARIA

J. A. ALLEN and M. R. GARRETT

Dove Marine Laboratory (University of Newcastle upon Tyne), Cullercoats, Northumberland

(Received 2July 1971)

Abstract-l. The taurine content of Mya arenariu maintained at full salinity varies considerably between individuals (3-13 mg/g dry wt.). There is an inverse relationship between size and taurine content of the tissue.

2. Taurine is lost when Myu is transferred to water of low salinity. How- ever, unlike Mytih which sheds almost all of its taurine when placed in sea water of .5x,, Mya maintains much ( > 50%) of its taurine at this salinity.

3. Mya can take up labelled taurine from solution at full salinity but little at low salinities. Tissue analysis and autoradiography show that most of the labelled sulphur is present in the gill and palp with smaller amounts in digestive gland and mantle. Injection of taurine and methionine resulted in a similar distribution.

4. Starved Myu having been kept at low salinities for 3 weeks and then returned stepwise to full salinity show no evidence of increase in taurine content.

INTRODUCTION

REMARKABLY little is known of the function of taurine in marine invertebrates, yet the evidence so far presented suggests that it is an important substance in many physiological processes (Allen & Garrett, 1971a). While taurine is present in exceptionally large quantities in marine bivalve molluscs, it is present in fresh- water bivalves in only small quantities. In euryhaline bivalves the quantity of taurine present is in direct relation to the salinity of the water (Allen, 1961). It has been suggested that taurine and free amino acids, such as alanine and glycine, are concerned in intracellular isomotic regulation and form part of the anion pools (Lange, 1963). Little is known as to how and where taurine is shed when the external salinity is reduced; however, in experiments on nitrogen excretion in Mya uwmz~ia, it has been shown that large amounts of ammonia are excreted under these conditions (Allen & Garrett, 1971b). Even less is known of the origin, or the rate of increase, of new taurine present in the tissues following the return to high salinities. These studies are an initial attempt to gain further information on the shedding and accumulation of taurine in the euryhaline bivalve M. arenuria.

307

308 J. A. ALLEN AND M. R. GARRETT

MATERIALS, METHODS AND RESULTS

Variations in taurine content of M. arenaria maintained at dz@rent salinities

Specimens of M. arenaria varying in length from 5.2 cm to 7.2 cm were kept in water of different salinities (4, 12 and 34x,) for periods between 27 and 64 days

at either 4 or 16°C. The taurine content of the soft parts was estimated in duplicate.

The soft parts were homogenized and then centrifuged at 4000 rev/min. Of the

supernatant, 5 ml was dried at 105°C and weighed, as was the precipitate. The

remaining supernatant was then passed through an 0.8 p Millipore filter and made

up to 10 ml with phthalate buffer and its taurine content estimated by the colori- metric method of Prentz et al. (1957), measurements being taken at 365 rnp using a Cambridge SP600 spectrophotometer and compared with a standard curve.

Some fifty-two specimens were analysed. The results show that there is consider-

able variation between individuals of a similar size and at the same salinity and temperature, e.g. 1.35-1.40 g body dry wt., at 16°C and 12x,, there is 5.7-8.7 mg

taurine/g dry wt. However, it is clear (Fig. 1) that there is on average, irrespective

of the salinity, a decrease in the quantity of taurine present per g dry tissue with

. .

.

.

0 . 0

.

I I I I I I I I I

OS ,I2 I6 2’0 24

Dry weight g

FIG. 1. Relationship of taurine content to total body dry weight in M. arenaria at different salinities and temperatures. A, 34x,; n , 127&; l , 47,, (outlined

symbols, 4°C; solid symbols, 16°C).

increase in size and weight of Mya. The estimations also show that the quantity of taurine differs little in animals kept at 12 and 34x0 salinity but at low salinities (4%,), while there is a reduction, considerable quantities of taurine remain-3-7 mg/g dry wt. (Fig. 1). This latter is in contrast to the findings of Lange (1963) for the euryhaline bivalve Mytilus who found that at 5x,, all taurine had been shed. Environmental temperature appears to have little effect on taurine content.

STUDIES ON TAURINE IN THE EURYHALINE BIVALVE ME’A ARENARIA 309

Taurine is present only in trace quantities in sea water. In earlier experiments

on nitrogen excretion in Mya, where animals had been maintained for several weeks in water of various salinities (Allen & Garrett, 1971b), analyses showed that taurine was not detectable in the water. Analyses also show that at the experimental salinities listed above, only trace quantities of taurine are present in the blood taken from the ventricle of the heart. This confirms a similar observation

for Mytilus by Lange (1963). It is assumed that intracellular taurine is metabolized

(probably via the intermediary isethionic acid, Allen & Garrett, 1971a) into ammonia and, possibly, sulphate ions.

Tissue analysis of two very large specimens of M’a, 10.6 cm and 10.1 cm shell

length, maintained at full salinity, showed that adductor muscle and pallial muscle had the smallest taurine content averaging 0.0063 g/g dry wt. and 0.0061 g/g dry

wt. respectively. The greatest quantity was recorded in the digestive gland, on

average 0.015 g/g dry wt., while ciliated mucus-secreting epithelial tissues such as gills and mantle to the inside of the pallial line contained intermediate amounts

averaging 0.0093 g/g dry wt. and 0.0106 g/g dry wt. respectively. Again this confirms observations on Mytilus by Lange (1963), although his observations giving the amount of taurine per wet weight tissue indicate that muscle contains

more taurine than gill or mantle in Myths. The discrepancy might be a reflection

of the vascularization of the tissues, indeed Lange (1963) thought that the variation

that he found could be explained by such differences. The present results indicate variation in taurine content of tissues may be real.

When euryhaline bivalves are returned to full salinity after a period in lowered salinity, it is likely that the new taurine necessary for ionic balance will be derived

from the food, possibly from the taurine or closely related taurine compounds known to be present in algae, but more likely via assimilated methionine (Allen &

Garrett, 1971a). While it is true that the low level of taurine content of sea water probably rules out direct uptake, it is possible that taurine could additionally be

synthesized from sulphate and ammonium ions. Thus, a few experiments were carried out to determine whether taurine in solution could be taken up by M. arenaria.

Mya measuring 5.7-5.9 cm total length were placed in aquaria holding 20 1. of membrane-filtered sea water (34x,,, 0.4~ porosity) to which taurine had been

added to give a range of concentration varying between 24 pg/l. and 38 pg/l. Analysis 24 and 48 hr after the introduction of the bivalves showed no significant drop in concentrations; however, after 16 days there was a loss of between 4 and 9 pg/l. (Table 1). Because the shells showed signs of sulphate deposition it was

clear that the loss could have been due to bacterial action. The experiments were therefore repeated using taurine 35S. However, before the uptake experiments were carried out, specimens of Mya to be used were kept in aquaria with 25 : 1 filtered sea water at the experimental salinity to which O-6 g of penicillin and 1.0 g streptomycin had been added. Eight days later the animals were transferred to fresh sea water with added antibiotics for a further period of 8 days. 0.12 PC taurine were added to experimental tanks containing 1 1. of filtered water of salinity 34 and


TABLE ~-THE UPTAKE BY M. arenatia OF TAURINE FROM SOLUTION IN SEA WATER (34x,) TAURINE CONTENT OF WATER GIVEN AS /.&g/l.

Taurine @g/l) content of aquaria

Day Control Mya 1 Mya 2 Mya 3 Mya 4

1 36.3 24.6 29.0 32.0 38.0 2 37.0 24.3 29.0 34.0 36.5

16 35.0 20.5 19.4 23.9 29.0

Length of Mya (cm) 5.74 5.72 5.92 5.97

10x0. A pair of Mya valves were added to a control tank at each salinity. Duplicate 0*2-ml samples were taken at various time intervals in the week following the start of the experiment and dried and counted with a G.M.4 end-window counter and Panax recorder. At the end of each experiment samples were taken of various tissues for counting and for autoradiography. After 7 days the Mya were transferred to fresh water of the correct salinity.

I01 , 1 , I I I I 1 I 2 3 4 5 6 7 8

Days

FIG. 2. Uptake of radioactive taurine by M. arenaria kept in sea water of 34x, (broken lines) and 10x0 (continuous lines).

The results (Fig. 2) show that at full salinity most of the s5S in solution is taken up from sea water within 48 hr by Mya; however, in the case of those at lowered salinity little is taken up by the bivalve. Analysis of the tissues of Mya at full salinity and 7 days from the commencement of the experiment show that the highest activity was in gill and palp tissue, with moderate activity in digestive gland and mantle. Little activity was recorded for gonad and adductor muscle (Table 2). In those Mya kept for 58 days after the commencement of the experiment, palp

STUDIESON TAURINE IN THEEURYHALI~BIVAL~ MYA ARENARIA 311

and gill still showed the greatest activity; similarly, mantle showed moderate activity and muscle showed little activity. However, in the case of the digestive gland there was a great reduction in activity and the gonad showed some increase in activity. &?“a kept at lowered salinity showed an entirely comparable picture 13 days after the start of the experiment to that of &ZJJG at full salinity 7 days after the start of the experiment.

TABLE ~--THE DISTRIBUTION OF 85S INTHE TISSUES OF THREE M. arenariu, FOLLOWING UPTAKEEXPERIMENTS USING TAURINS 3 IN SOLUTION (countsjmg dry tissue per min)

Countsfmg dry tissue per min

Tissue Mya 1 Mya 2 Mya 3

Mantle epithelium Pallial muscle Adductor muscle Palp Gill Gonad Digestive gland Style

469 210

24 3132 3265

167 697

3

246 73 49

960 668 167

85 -

103 63 38

239 526 154 261

4

Time from start of experiment (days) 6 58 13 Length of Mya (cm) 5.69 5.58 5.21 Salinity 34%0 34%0 lo%0

Experiments involving the injection of radioactive taurine and methionine

The level of activity within the tissues of Mya 7 weeks after uptake of taurine “S from solution is very similar to that found 1 week after the start of the experiment. This would indicate that s5S, at least, is retained in the body even though taurine may be degraded. In order to investigate this further, Mya, which had been kept in filtered sea water to which penicillin and streptomycin had been added, were injected with O-1 ml taurine solution containing 0.012 PC taurine 955. Injection was through the posterior adductor into the kidney/heart region, After injection, the specimens were washed and placed in aquaria containing 500 ml of membrane filtered sea water. The water was monitored daiIy until the end of the experiment, after which a tissue analysis was carried out. Results, 7 and 40 days after injection, show that there is no rapid loss of taurine. Count rates of the water indicate a very low level of activity-just above background-with no indication of any build-up of the level.

Tissue anaIysis after a week (Table 3) shows that, as before, the gin has by far the highest count rate of the tissues anaIysed. Palp, gonad and digestive gland also contain appreciable quantities. The distribution after 40 days is essentially the same as after 7 days.

Methionine is generally regarded as the starting point in taurine metabolism (Allen & Garrett, 1971a). Specimens of M. arenarik were injected with O-04 mc


TABLE 3-DISTRIBUTION OF 35S IN THE TISSUES OF M. Urf?TZUriU FOLLOWING INJECTION

OF TAURINE YS (counts/mg dry tissue per min)

Counts/mg dry tissue/min __~ ___ ____

Tissue Mya 1 Mya 2 Mya 3 ____--

Mantle epithelium 7 28 27

Pallial muscle 4 4 5

Adductor muscle 4 3 6

Palp 21 11 53

Gill 137 48 185

Gonad 23 15 47

Digestive gland 21 10 14

Style 0

Time since injection (days) 7 55 56

Length of Mya (cm) 5.11 5.26 5.14

35S methionine in aqueous solution into the body anterior and posterior to the

ligament. After injection the animals were kept in filtered sea water at 10°C for intervals of 1, 6, 12 and 40 hr, after which a small portion of the gill and mantle

was removed from each specimen and fixed for autoradiography. The remainder

of the soft parts were homogenized and the free amino acids and taurine extracted

and chromatogrammed. The method of analysis used was a modified version of that of Awapara (1948). Exchange resin Deacidite FF was recycled six times before forming 12-cm columns and, once formed, the columns were recycled a further five times. The homogenized soft parts were deproteinized in 80% ethanol. The

supernatant was diluted to 250 ml with distilled water and desalted by passing

90 -

SO-

70-

60-

LL I 50- tl

f 40-

30 -

201.

IO-

8 6hr

A B C D E F B C D E

FIG. 3. Two examples of chromatogram scans of extracts from 2M. aremaria, prepared 6 hr and 1 hr following the injection of 35S Methionine. Peak C, methio-

nine; peak E, taurine; peak F, cysteic acid.

STUDIES ON TAURINE IN THE EURYHALINE BIVALVE MYA ARENARIA 313

through the column in the basic phase. After washing the column was extracted by 2N HCI and the effluent was evaporated to dryness in a rotary film vacuum evaporator at 40°C and recrystallized by repetition. The crystals were then dis- solved in a minimum quantity of 10% isopropyl alcohol and chromatographed on paper using 5 ~1 of the solution, and run with N butanol-acetic-water (450 : 50 : 125 v/v), dried and rerun, and then developed in 0.25% ninhydrin to each 100 ml of which one drop of pyridene had been added. This was compared against standard sulphur ammo-acid solutions using a Panax continuous scanner.

Six peaks (A-F) were present in the chromatograms (Fig. 3) and of these C, E and F correspond to methionine, taurine and cysteic acid respectively. The others, A, B and D are unknown. Relative values of deflection for these six peaks are given (Table 4) using the 10 per cent level as the base line (Fig. 3). The results from each experimental run are very similar.

TABLE ~-PEAK DISTRIBUTION, R, AND SIZE AT THE VARIOUS TIMES FOLLOWING INJECTION

OF M. UYt%UY&2 WITH “s METHIONINE

1st series 2nd series

Time Deflection Time Deflection

(hr) Peaks R,x 100 (%) (hr) Peaks R,xlOO (%I

1 B 70 10 1

Methionine 50 85

D 35 50

Taurine 11 100+

6 A B

Methionine D

Taurine

Cysteic acid

15 A B

Methionine

D Taurine

Cysteic acid

40 Methionine D

Taurine

Cysteic acid

93 20 6

78 40

52 3.5

38 25

13 85

4 60

95 5 15

88 7-5

60 25

40 40

15 100 9 80

62 20 40

39 30

17 55

3 100+ + +

A

B

Methionine

D

Taurine

Cysteic acid

A B

Methionine D

Taurine

Cysteic acid

Methionine

D

Taurine

Cysteic acid

Methionine 49 20 D 34 1.5 Taurine 10 40 Cysteic acid 1 lOO+

78 14

66 14 49 60

3.5 25 11 50

1 20

75 13 66 7 49 20 35 15

11 30 2 35

48 20 34 20

7 65 2 50

In general, the experiments show a quick turnover of methionine, well over half that injected being converted into other products within the first hour. The


methionine peak diminishes to become more or less a constant value after 15 hr. Taurine and peak D reach their maxima between 1 and 3 hr then decline again possibly D being somewhat in advance of taurine. A and B reach maxima at about 5 hr and are not recorded after 15 hr, while in the case of cysteic acid, there is an initial fast rise in the first 5 hr followed by a further more gradual rise up to 40 hr. The main point of interest is that the 35S is not retained in taurine but is passed relatively quickly along the pathway(s). It should be noted that (1) up to the time of the experiment the specimens of Mya were feeding and (2) the experiment was completed in under 2 days. However, it would seem that up to 50 per cent of the methionine 3sS is converted to taurine 3sS within the first hour. It may well be that these rates are a reflection of the disturbance that excess of methionine gives to the balance of the amino acid pool although the amount of dietary methionine must itself be variable.

From the first series of experiments it was clear that Mya does not lose so much taurine at low salinities as does Mytilus (Lange, 1963). This may be but a reflection of the relative ability of the two animals to regulate. However, what is not clear in either case is whether, on return to higher salinity, the normal taurine level is re-established quickly, and whether re-establishment is from synthesis of existing precursors possibly involving uptake of ions from sea water, or whether an increase in taurine content is dependent on incoming food materials.

Twelve Mya were acclimatized to low salinity (lo%,) and starved over a period of 3 weeks in filtered sea water. Two were then analysed for their taurine content and the remainder placed in filtered sea water of a higher salinity for a period of 4 days, after which a further two specimens were analysed and the remainder transferred to yet another higher salinity. The procedure was repeated until full salinity was reached (Table 5). Th e experiment was then repeated a second time.

Results indicate that there is no increase in the taurine content in these animals, rather the reverse is indicated, with falling taurine content with increasing salinity. This is pa~icularly clear in the first series. The results also show again clearly that the smaller the specimens the higher the taurine content/g dry tissue. It must be concluded that starved animals in low salinities are not able to raise quickly the level of their taurine content on return to higher salinities. It has been shown that in vertebrates taurine can be synthesized via sulphate ions (Allen & Garrett, 1971a) ; however, it is likely that this is not a major pathway and it would appear that this is true also of My@.

Autoradiography

In all uptake and injections experiments, tissue was fixed for autoradiographic processing. In general these sections confirm the results of the tissue analysis. In Myu injected with radioactive methionine and taurine, activity is widespread

STUDIES ON TAURINS IN THE EURYHALINB BIVALVE MYA ARENARIA 315

TABLE 5-TAURINE CONTENT OF M. arenaria STARVED AT LOW SALINITY, FOLLOWED BY

A STEP WISE INCREASE IN SALINITY

Time Salinity Shell length Dry wt. Taurine

(days) (%o) (cm) (g) (mg/g)

Experiment 1

21 10

21 10

4 14

4 14

2 18

2 18

4 22

4 22

2 26

2 26

3 30

3 30

Experiment 2

21 10

21 10

2 15

2 15

4 21

4 21 2 24 2 24 5 34

6.8 1.73 7.2 2.22 6.2 1.55 7.0 1.64 6.9 1.51 7.0 1.80 6.9 1.90 7.0 1.93 7.0 2.25 7.1 2.41 6.2 l-32 7.9 2.35

6.6 6.3 6.4 5.9 5.5 6.9 6.3 6.1 6.3

l-59 1.36 1.77 1.48 1.01

2.05

1.76

1.52

1.77 2.4 2.4

within the tissues whereas in uptake experiments it is more peripheral in its distribution. However, they all show one thing in common, that there is a concentration of labelled sulphur in those tissues which secrete quantities of mucus. The gills are a particular case in point with concentrations in the frontal cells and in the mucus cells posterior to the lateral cilia. Similar results are obtained in uptake experiments with 35S04 ions (personal observations). It should also be pointed out that in many sections the mucus overlying the epithelia (e.g. frontal surfaces of the gills) was strongly labelled.

DISCUSSION

The experiments and observations described highlight a number of points of interest in regard to taurine in M. a~enu~iu. The concentration of many substances within an animals’s body varies in different individuals and for various reasons even within the same individual and, as such, the degree of individual variation in taurine is not exceptional. However, reduced taurine concentration with increasing size (age) of the animal is unexpected and not easy to explain if taurine is acting, in the main, in intracellular isosmotic regulation (Lange, 1963). If the


latter function was paramount then one might expect that there would be a direct relationship between taurine and the amount of body tissue. However, it is clear that osmotic function is not the only one in which taurine is concerned (Allen & Garrett, 1971a), and that taurine, an intermediate in sulphur amino-acid metabolism, may possibly be concerned with the formation of sulphonated mucopoly- saccharides, that is, it provides the sulphur content of the mucus that is so essential to bivalve function. To support this idea there are the following facts. Molluscs, which are particularly rich producers of mucus, have a high taurine content, conversely, crustaceans which produce much less mucus have a low taurine content. Furthermore, autoradiography of animals injected with taurine or methionine or that have been the subject of uptake experiments shows high concentrations in those epithelia that produce the most mucus (gills and palps). Mucous glands frequently show concentrations of labelled sulphur and the mucus film over these surfaces is also richly labelled. If this is a function of taurine in the bivalve, then it might explain the drop in taurine concentration with increasing size, in that it is area of epithelium and not volume of animal that is significant. In fact total amount of taurine present increases with increasing size (approximately l-5 mglg increase in wt.).

Mjla differs from Mytilus (Lange, 1963) in that although taurine is shed when these animals are transferred to water of lower salinity, the loss is not nearly so great in Mya. Thus, in MJXZ there is relatively small change in the taurine at 12%0 and, although there is a clear reduction at 4x,, considerable quantities of taurine remain in the tissues. In the case of Myt&s edulis almost all the taurine is shed at 4x,. It seems that there is little osmotic regulation in Myte’lus (Robertson, 1964),

80-

.

So’, , , , , ( , , , , , , ,

08 12 16 20 24 28 32

Dry weight g

FICL 4. Relationship between shelf length of M. arenaria and the dry weight of its soft parts.

STUDIES ON TAURINE IN THE EURYHALINE BIVALVE MYA ARENARIA 317

unfortunately little is known of osmotic regulation in Mya but the present evidence

might suggest that it occurs. Although Mya can readily take up taurine from solution at full salinity, the

fact that so little taurine is present in sea water precludes this as being a major source of supply. A series of experiments in which starved animals at low salinities

were subject to a stepwise increase in salinity, and which showed no evidence of an increase in taurine content, suggests that any increase in the taurine content

must come via the food. In fact experiments involving the injection of methionine

suggest that metabolism of the source amino-acid is rapid and may well provide the necessary increase in taurine level.

ADDENDUM

The experiments have provided useful information on the relationship between

shell size and soft body dry weight in Mya (Fig. 4). Few results are included in the literature on this relationship. No account is taken of the state of maturity of

the animal.

Acknowledgements-We wish to thank the Science Research Council for their support through Grant No. 17762.

REFERENCES

ALLEN J. A. & GARRETT M. R. (1971a) Taurine in marine invertebrates. Adv. mar. Biol. 9, 205-253.

ALLEN J. A. & GARRETT M. R. (197lb) The excretion of ammonia and urea by Mya arenariu

L. (Mollusca: Bivalvia) Corn@. Biochem. Physiol. 39, 633-642. ALLEN K. (1961) The effect of salinity on the amino acid concentration in Rungiu cuneutu

(Pelecypoda). Biol. Bull. mar. biol. Lab., Woods Hole 121, 419-424. AWAPARA J. (1948) Application of paper chromatography to the estimation of free amino

acids in tissues. Archs Biochem. 19, 172-173. LANGE R. (1963) The osmotic function of amino acids and taurine in the mussel, Mytilus

edulis. Comp. Biochem. Physiol. 10, 173-179. PRENTZ E. I., DAVENPORT C. H., GLOVBR W. & SMITH D. D. (1957) A test for the deter-

mination of taurine in urine. J. biol. Chem. 228, 43346 ROBERTSON J. D. (1964) Osmotic and ionic regulation. In Physiology of Molluscu (Edited

by WILBER K. M. & YONGE C. M.), Vol. 1, pp. 283-311. Academic Press, New York.

Key Word Index-Taurine; Myu arenuriu; osmoregulation; salinity.

studies on taurine in the euryhaline bivalve mya arenaria

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