structure and function of crystalline styles of bivalves

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STRUCTURE AND FUNCTION ... Rotation of the style is central in discussions on the function of bivalve stomachs, although estimates of .... 0 • ______. 0 _ •. 0.
OPHELIA, 10: 91-108 (August 1972)

STRUCTURE OF CRYSTALLINE

AND FUNCTION STYLES OF BIVALVES

J. HYLLEBERG

KRISTENSEN

Zoological Institute, Lab. B, Ecology, University of Aarhus, DK-8000 Århus C, Denmark

ABSTRACT The general appearance of crystalline styles is described. Experiments show that styles are renewed every 4th hr in Abra nitida and about every 24th hr in Macoma balthica . Styles are not simply dissolved from the anterior end as previously assumed, material from the center of the styles is expelled also. Some physical properties of style solutions are described : the surface tension is decreased, oil is efficiently emulsified, the viscosity is increased and pH buffered. These properties and enzymatic activity of style material are discussed in relation to the generally accepted functions of crystalline styles . Intermittent rotation of styles and an observed effect of hepatopancreas extract in increasing the dissolution of whole styles are discussed. It is concluded that style material is of importance in the sorting of food partides.

INTRODUCTION Crystalline styles are transparent rods found in the digestive system of certain gastropods and all bivalves except protobranchs, which have a so called protostyle. Styles are found either in a separate style sac without direct contact between style and intestine, or there is a slit in the sac so that sac and intestine (mid gut) are incompletely separated, or the style may lie in the intestine itself. The style projects from its sac into the stomach where it is generally believed to press against the gastric shield. During rotation the head of the style is claimed to be worn down by grinding against the surface of the shield (Yonge, 1923, 1949). Rotation of the style is central in discussions on the function of bivalve stomachs, although estimates of the speed of rotation vary within wide limits (Nelson, 1918; Yonge, 1949; Dinamani, 1957; Reid & Reid, 1969; Morton, 1970). Rotation of the style is supposed to cause mucus bound material to be drawn from the oesophagus of the tip of the style where the material is stirred and mixed with enzymes. Stomach and style are believed to aet together as an organ of trituration (Y onge, 1949). Crystalline styles are known to contain enzymes, especially carbohydrases (Owen, 1966; Kristensen, 1972) though the actual amount of enzyme protein appears negligible in terms of weight (Bailey & Worboys, 1960). This observa-

92

J. HYLLEBERG

KRISTENSEN

tion, together with my experiments on style enzymes, indicates that the weak enzymatic activity is hardly in accordance with the generally accepted view that styles are worn down in order to mix food particles with enzymes in extracellular digestion. The present observations on the morphology of styles especially in the tellinacean .Macoma balthica are not in agreement with descriptions of tellinaceans given by Yonge (1949). The purpose of this paper is to describe these differences as well as experiments on hitherto undescribed properties of crystalline styles which are considered important in the digestive processes of bivalves. Financial support from Statens Naturvidenskabelige Forskningsråd and working facilities at the Kristineberg Zoological Station and the Marine Biological Laboratory, Helsingør are gratefully acknowledged . My sincere gratitude is due to my colleagues Drs. K. Ockelmann, T. Fenchel and stud.mag. scient. B. Barker Jørgensen for valuable discussions, and to Miss E. Glob for help in all analytical work.

MATERIAL

AND METHODS

The bivalves Macoma balthica (L.), Mya arenaria L., Mytilus edulis L., and Cardium edule L. were collected at the brackish-water localities Nivå Bay (northern part of Øresund) and Kysing Fjord (east coast of Jutland). The animals were placed in aerated sea water in a constant temperature room at 8 ° C or in the laboratory at room temperature. Abra alba S. Wood and Abra nitida Muller were collected at the west coast of Sweden and kept in running sea water at 18° C at the Kristineberg Zoological Station.

RESULTS Morphology of Macoma balthica styles

Figs 1-2 show the general appearance of a freshly dissected style. Styles appear as hyaline rods rounded posteriorly and drawn into an elongated tip at the anterior end. The following characteristics are indicated in Fig. 1: a) A lump of fine detrital particles, diatoms, bacteria and minute quartz grains imbedded in sticky mucus. b) The tip of the style which is often twisted and projects into the stomach (Fig. 5B). c) Very fine particles embedded in sticky mucus at the surface of the anterior part of the style. d) A more or less bulbous part set off from the rest of the rod by e) a collar of often very marked impressions in the style material. f) Coarse lamellae which are curled in the bulb and curved to a variable degree in the rest of the style. Newly added lamellae are elevated 5-10° to the longitudinal axis of the style while lamellae near the axis describe a sigmoid curve. Lamellae are usually close to each other at the collar w hile more spaced towards

CR YSTALLINE a

b

C

d e

STYLES

93

OF BIVALVES

f

posterior

anterior

1mm

FIG. 1. Semischematic drawing of the crystalline style extracted from Macoma balthica. See text for legend .

the centre of the style. The coarse lamellae are composed of finer lamellae visible only at higher magnifications (Fig. 2). g) A central core of granular material. The nearby systems of lamellae disappear gradually in the granular zone as seen in Fig . 2. The granular zone continues to the exterior. Invaginated lamellae of the outmost layer are observed in many styles (Fig. 2, h). The invagination extends as a "chimney pipe" when the style dissolves in vitro.

g

f

h

300 µ FIG. 2. Posterior style section (phase contrast, 100 X). f, Fine lamellae. g, Granular central core. h, Invagination of lamellae observed in many styles.

94

J. HYLLEBERG

KRISTENSEN

mm 4. 00

sea water

p H = 5. 9

3.50

3 00

2.50 Lengt

h of st yles

Wld t h o f styles

2.00

I.SO

1.00

_ _ _ _ _ 0 • _______

0 • _______

0 _ •

0

''

0.50

0+-------~------~-----.10-----,--------~-o

'''

''

0

'

- ____

-'0

I. ho u r s

FIG. 3. The speed of style dissolution with and without addition of hepatopancreas extract. Length and width of styles remain fairly constant until styles suddenly collapse. See also Fig. 4.

In vitro dissolution of crystalline styles

Liquefaction of styles has hitherto been explained as a simple dissolving of style protein, the rate of which depends only on the pH of the medium (Yonge, 1930) and temperature (Berkeley, 1959). A pH dependence in the dissolution of styles of Macoma balthica was also found. At pH 3.5-4.0 (adjusted with HCl) styles remained solid for days at room temperature. With increasing pH, styles dissolve more and more rapidly , e.g. in about 1 hour at pH 7.8. At pH 9.0 (adjusted with NaOH) Macoma styles elongate and dissolve completely in less than 1 hour. Styles placed in phosphate buffer dissolve even faster at this pH. They elongate vigorously and whole systems of lamellae are repeatedly expelled at the anterior end of the style. With citric acid added to the sea water, styles dissolve at values of pH at which they are insoluble or dissolve only slowly with HCl-adjusted pH. Dissolution of crystalline styles in two ml sea water is very effectivelyprevented by the addition of a few crystals of ammonium molybdate which forms (NH 4 ) 3 P0 4 , 12 Mo0 3 with the phosphate present in the styles (Doyle, 1966). Precipitation of cations in the medium by potassium oxalate has no effect on the dissolution of Macoma styles.

CRYSTALLINE

STYLES

95

OF BIVALVES

t = 2min.

2

t = 25 min.

3

t=80min.

4

t = 115 min.

5

t = 135 min.

6

t = 160 min.

-----:::::===~----',

..--:;:::::;=---7

---,

___

I

t=17 0 min .

---.;,.....U-/.,' ("- .... -,....... , ,.,

...._

\

I

I I

,,

I

8

II

' I I \

\

I

'\

t = 195 min .

\ /' I I

'--- ...._,.,.._.,,

1mm

FIG. 4. Stages in dissolution of a Macoma balthica style (25 X ). Arrows indicate alteration in position of a certain lamella during dissolving of the style. Dotted contours indicate poor contrast of the style material expelled.

96

J. HYLLEBERG

KRISTENSEN

Yonge (1926) studied the effect of hepatopancreas extract on style dissolution in Mya arenaria. He found that addition of fresh extract accelerated the dissolution while boiled extract had little or no effect. My experiments with fresh hepatopancreas extract (Macoma) are in agreement with Yonge's findings (Fig.3). The substance causing this effect is unknown. Destruction of the substance by boiling indicates that enzymes might be involved. This view, however, was rejected by Yonge (1930). Fig. 4 shows various stages in the dissolution of a Macoma style in 2 ml sea water (pH adjusted with HCl) after addition of hepatopancreas extract from the same individual. It should be noted that addition of style sac extract has only little effect on dissolution compared with styles in pure sea water. When removed from the style sac, the length of the style slightly increases (Fig. 4). The section anterior to the collar (Fig. 1) swells and dissolves slowly. A little of the granular material is expelled at the posterior end while the systems of lamellae close to the central core swell and elongate. This style material is expelled anteriorly, often as a whole system of lamellae, and dissolves in the medium (arrows in Fig. 4). Due to contraction and dissolving of style material the style gradually dissappears from behind as seen in Fig. 4 but it keeps the rod form and diameter until the final collapse.

In vivo formation and dissolution

The style is secreted from the style sac (Kato & Kubomura, 1954) and according to Y onge (1949) pushed forward, slowly dissolving in the less acid medium of the stomach. However, the elongated style tip with its lump of detritus (Fig. 5B) and the in vitro observations described above are not in accordance with this view. In order to study the formation and dissolution of the crystalline style in situ, bivalves were placed in sea water stained with trypan blue (BDH). As a vital stain this high-molecular, acid dye accumulates in cell vacuoles or it stains proteins diffusely. In Macoma balthica and Abra nitida the style and parts of the gastric shield, especially the dorsal tooth (Fig. 5 B) were stained diffusely. After 12-18 hrs in sea water with trypan blue (about 0.01 %) styles appeared quite blue. Bivalves were then transferred to aquaria with sediment and unstained sea water, and dissected at various intervals (Fig. 6). Natural sediment and aeration of the aquarium is necessary to obtain a successful staining, otherwise styles remain unstained. Apparently no dye is incorporated into styles unless animals are feeding. Macoma dissected after 4 hrs in unstained sea water, had a thin layer of hyaline unstained material added to the style along the part found in the style sac (Fig. 6). After 24 hrs the thickness of new material significantly increased in 13 individuals while 2 other styles were totally hyaline, indicating complete renewal. After 7 days blue material still formed a small central core in 1 of 5 individuals.

CRYSTALLINE Dorsal

STYLES

OF BIVALVES

97

hood

1mm Crystall

ine style

B

FIG. 5. A, Stomach and crystalline style sac of Macoma balthica . Arrows indicate movements of partides in the stomach. B, Stomach and style sac cut open to show position of the crystalline style. Arrows indicate movements of partides around the left pouch.

Abra nitida renewed styles much quicker than Macoma balthica, perhaps due to more natural conditions of recovery in unfiltered, running sea water. After 4 hrs, 15 Abra nitida individuals studied had renewed 90 % of the style material (see Fig. 6). When stained styles dissolve in sea water, most of the blue material forming the central core is expelled at the anterior end and only a little posteriorly. Summarizing, trypan blue staining indicated an addition of new style material along the whole section situated in the style sac, although mainly at the posterior end. The "old" style material is forced towards the centre of the style from where it gradually enters the stomach.

Properties of dissolved styles Surface tension: It is well known that proteins and many other organic compounds reduce the surface tension of water. This property was studied on style solutions by a capillary tube technique. The height of the column of a solution of 6 mm styles of Macoma balthica dissolved in 50 µl sea water was measured to the nearest mm in a capillary (radius = 0.3 mm). The decrease in surface tension compared to sea water was 22, 22, 20, and 21 % respectively. The capillary was rinsed with "cleaning mixture" between each of the four exp~riments. This decrease corresponds with a calculated surface tension of about 56 dyn/cm (surface tension = ½hdgr; where h is height, d density, g constant of gravity, and r radius). This decrease was expected, as half of the organic style matter

98

J. HYLLEBERG

KRISTENSEN

A

t =4h

B

t =1 day

C

t = 7 days

D

t =4h

1 mm Fm. 6. Styles of Macoma balthica (A, B, C) and Abra nitida (D) stained in vivo with trypan blue in sea water. Granular areas show tr ypan blue material still visible after the indic ated periods in unstained sea water. Hy aline areas show addition of new style material (lamellae). The tuft at the style tip (D) consists of unstained detritus partides.

is protein (Doyle; 1966). Hepatopancreas extract of the same species decreased surface tension about 46 % and style sac extract about 17 %Emulsifying property: Style material is a very effective emulsifier. Fig. 7 shows the emulsification obtained of a drop of olive oil in sea water, when freeze dried Cardium edule style is added. Optical density (Beckman B spectrophotometer , 1 cm cells) was measured after shaking of the photometer cell. The emulsion was still milky after 24 hrs. The emulsifying property may be related to the decreased surface tension. Viscosity: The viscosity was studied in an Ostwald viscosimeter. Freeze dried Cardium edule style material was dissolved in Phosphate-citrate buffer in sea water at pH's 4.8, 5.8 and 6.9. The relative viscosity calculated by the expression "YJ

sp = -

't

'ro

(--r and

--ro are

the readings in seconds with and without style material

respectively), showed a linear increase in viscosity with increasing pH (Fig. 8).

CRYSTALLINE

STYLES

99

OF BIVALVES

Optical density at 650 nm

10.0

0.1 mg style

extract

added

5.0

Olive

5

oil

in sea water

10

15

min.

Fm. 7. Emulsifying property of style extract (Cardium edule) shown by increase in optical density after addition of style material to photometer cell with olive oil in sea water (4 ml).

The style protein was precipitated with (NH 4) 2 SO4 at increasing concentrations (25, 50, 75 and 100 % saturation). At 25 % saturation a slight turbidity appeared but only the precipitate formed at 50 % could be centrifuged and collected. The 75 and 100 % saturations did not give precipitates (pH 6.5). The centrifuged precipitate and the material remaining in solution were dialysed against tap water for 24 and 48 hrs respectively, freeze dried and weighed on a microbalance. 3.5 % (1.4 mg) of the material was lost by this treatment. On a weight basis the proportion of globulin-like material (precipitate) to the remaining

100

J. HYLLEBERG 'h sp =

l

KRISTENSEN

'r 'to

1.117

1.097

1.077

+ 1.05 7

C:,

__s-

2 .5 mg precipitated with ( NH 4 J2 so 4

____s:-7.5 mg

i

i

i

4.8

5.8

6.8

not

precipitated

' 7.8

pH

Fm. 8. Relative viscosity of freeze dried Cardium edule style material measured in an Ostwald viscosimeter (25 ° C). Whole style extracts and fractions obtained by precipitation with ammo nium sulfate were measured. Each value represents average of 10 measurements. The indicated amounts of material were dissolved in 10 ml buffer at the indicated pH's.

material was 1.04 to 2.85. The viscosity of the two fractions was measured. It is likely that the precipitated material contributes more to the observed viscosity than does the unprecipitated style matter. A reservation is made as the latter fraction would not dissolve completely in the buffer. The effect of viscosity on natura! detritus was studied photometrically by using the sinking rate of partides less than 125 µ (Fig. 9). It is obvious that small partides are kept in suspension for a longer period of time when the viscosity (and also density) of the medium is increased. Enzymes: It has long been known that crystalline styles contain enzymes, especially carbohydrases. The activity of carbohydrases in 0. 7 mg freeze dried extracts of hepatopancreas and crystalline styles from Mya arenaria was compared.

CRYSTALLINE

STYLES

101

OF BIVALVES

Optical density at 650 nm

1.00

0.90

0.80

0 .70

0.60

0.50

atter addition

0.40

of style

material

o/

0.30

0.20

0.10

0 -t-----,,----,-,---"T---r---,--.--~--,---,-----,--,--.---~-~0 2 3 4 5 6 7

8

9

10

11

12

13

14

15 min

FIG. 9. Sinking rate of natural detritus before and after addition of 3 mg freeze dried Cardium edule style material to 3 ml sea water. Curves show average of 3 measurements, vertical bars indicate range of measurements.

The method is described in detail in Kristensen (1972). Fig. 10 shows the increase in reducing sugar when extracts are incubated with amylose, glycogen and laminaran. RS (reducing sugar) values were estimated after 1 and 4 hrs respectively. It is seen that hepatopancreas extract is the most effective in hydrolysing carbohydrates at the pH used. Similar evidence was found by Crosby & Reid (1971). The ratios of hydrolysis (Fig. 10) are calculated as 9: l in amylose, 4: 1 in glycogen and 6: 1 in laminaran after 4 hrs.

102

J. HYLLEBERG



µg RS 500

D

C

KRISTENSEN

hepatopancreas C rystalline

style

extract extract ~

100 -

~

500

100

-

-

-

-

10-

-

5 _....__ AMYLOSE

4

...10

.___

GLYCOGEN

4

4

5

LAM INARAN

4

4

4

hou rs

Fm. 10. Amounts of reducing sugar (RS) measured photometrically after incubation of 0.5 mg polysaccharide with 0.7 mg freeze dried Mya arenaria extract in 100 µ1 buffer pH 5.6 at 32°C.

Qualitative tests (chromatography) showed that maltase, laminarase and amylase present in crystalline style preparations of Macoma balthica also were present in style sac preparations, including the attached mid gut. Details on spectra of other style carbohydrases are published separately (Kristensen, 1972). Action on microorganisms: Johansson (1945) observed that extracts of Mya styles remained clear for weeks even when inoculated with bacteria. This effect disappeared in boiled and ethanol treated extracts, indicating the presence of a bacteriotoxic, thermolabile substance (oxidase?) in fresh styles. Dean (1958)

CRYSTALLINE

STYLES

103

OF BIVALVES

Optical density at 650 nm

+-+ 6 -

0 .400

o-o

without 6

addition

2 mg style

material

3 crystalline

styles

0.300

0 .200

0

2

3

4

5 hours

Fm. 11. Alteration in optical density of bacteria cultures in 3 ml peptone-sea water. Addition of freeze dried style material, or fresh styles of Cardium edule have a restraining effect on the multiplication of the bacteria.

found that dissolving styles disintegrated Cryptomonas cells and immobilized Monochrysis while Isochrysis was unaffected. Studies on Isochrysis incubated with 6 mm whole styles of Macoma balthica at room temperature show that swimming stops after about 1 hour. In accordance with Dean (op. cit.) flagellates do not disintegrate. However, after 24 hrs

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J. HYLLEBERG

KRISTENSEN

nearly all Jsochrysis were dead and the lumps of algae served as substrate for bacteria. Comparison with untreated controls showed 100 x more bacteria in tubes with styles and dead lsochrysis. Although the origin of the bacteria is unknown the observation seems contrary to the tindings of Johansson (1945). Some experiments were, therefore, set up to study the effect of style material on bacteria. A dense culture was prepared by the inoculation of natural detritus in sea water with 0.1 % pepton. One ml of this suspension was mixed in photometer cells with sea water and 2 mg freeze dried Cardium edule styles, or with sea water and 3 fresh styles. The cells were incubated (tight titting stopper) and the optical density read at intervalsafter shaking of the contents (Fig. 11). Similar experiments with Macoma balthica styles and with phosphate-citrate buffer pH 6.8 instead of sea water showed that addition of fresh styles as well as freeze dried material has an inhibiting effect on the multiplication of the inoculated bacteria, although the effect does not seem pronounced in buffer solutions. Microorganisms occurring in material adhering to styles of freshly collected bivalves (Mya, Cardium, Macoma) were studied under a fluorescense microscope after vital staining with acridine orange (Fenchel, 1972). The material always contains living bacteria and diatoms, indicating that microorganisms are not immediately killed at the tip of dissolving styles. This is in accordance with Reid (1969) and Fenchel (1972). The latter author found high bacterial numbers in the stomach of Macoma while bacterial numbers were signiticantly decreased in the intestine, indicating lysis and digestion of microorganisms in hepatopancreas rather than by style extract in the stomach . Experiments on the oxygen uptake of detritus with and without style extracts added did not indicate any effect on microbial respiration.

DISCUSSION The presence or absence of crystalline styles in living bivalves has often been discussed. Berkeley (1923) found that the absence of styles was a direct response to lack of oxygen in the environment. However, there is no evidence to support this view (Yonge, 1926). Yonge (1923) concluded that the presence of styles is dependent solely on the type of style sac, i. e. styles are always present if style sac and intestine are completely separated . He studied Mya arenaria, and I also found styles to be extremely persistent in this species. In addition they are difticult to dissolve in water compared with the styles of Cardium, Macom a and Abra. However, the in vivo experiments with staining of crystalline styles show that formation and dissolution of styles only take place when bivalves are feeding a tinding which coincides with Morton (1970) and various authors cited by

CRYSTALLINE Shell mm

STYLES

105

OF BIVALVES

len g th

15

14

• = Abra

nitide

(y= -0 05+2 .90x)

13

12

11 Abra

alba

=

O

(y=2 .99+1.97x)

10



0 0

9

8

St yl e length I 3

I

I

I

4

5

6

mm

FIG. 12. Crystalline style lengths plotted against shell lengths in Abra nitida and Abra alba. Calculated regressions are shown.

106

J. HYLLEBERG

KRISTENSEN

Yonge (1923). This is also supported by my own dissections of several hundred tellinaceans which always contained styles irrespective the time of the year, although styles may be somewhat soft in winter and in starved animals. Fig. 12 shows the relationship between style and animal size in two tellinacean species kept in aquaria with natura! sediment. Styles were present in all individuals and measured about 1/3 of the shell length. A very important point regarding the function of the style is that it rotates vigorously (Yonge, 1949; Owen, 1953). However, Owen never - and Yonge only once - saw a rotating style. I have looked intensively for rotating styles without success. Styles cut free from the style sac may revolve for a short while due to untwisting of the twisted style tip (Fig. 5B). Nevertheless, styles must be assumed to rotate due to the uniform addition of lamellae in bivalves without a separate style sac. An intermittent rotation is proposed by Morton (1969, 1970) and Purchon (1971) but details are not given. Johansson (1940) gave evidence of a nervous control (inhibition) of cilia in the style sac of Mya arenaria. This observation has to my knowledge not been discussed in the literature but I consider it important as nervous control could be the mechanism regulating an intermittent style rotation. It is an intention of future research to show how addition of new style material is coordinated with style rotation. It seems justified to discuss crystalline style function in terms of stored material which can be drawn upon when needed, but a mechanism must then be suggested which regulates the release of style material at the appropriate time. Unfortunately nothing can be said as regards this regulating mechanism. Proteolytic enzymes and pH differences in stomach and style sac have been proposed. However, it should be noted that an increased uptake of water by the style material would result in squeezing out of style material as observed in vitro in this study. Contrary to whole styles a swelling was observed in longitudinally sectioned Macoma styles, indicating a difference in water absorption of the two sides of style lamellae, i. e. hygroscopic towards the central core. In addition diffusion of ions was found to be faster through the central core than through the remainder of the style (styles immersed in silver nitrate in dist. water for a short time and then developed in a photographic developer were black at the outside and in the central core, while the remaining style matter was brown). According to my dissections, the bulb of the style is placed in the stomach (Fig. 5 B), but it is difficult to judge the exact position due to contraction of the individuals. My suggestion lies between those offered by Yonge (1949) and Reid (1969), the former placing the bulb (of Tellina tenuis) in the dorsal pouch and the latter placing the whole style (of Macoma secta) in the style sac and the tip with its lump of detritus in the stomach. Y onge (op. cit.) explains the position and the size of Tellina styles as an adaptation to unselective feeding. He

CR YSTALLINE

STYLES

OF BIVALVES

107

found an abrasion of sand grains in the stomach due to trituration between style and gastric shield resulting in reduction of the size of partides of sand from 320 µ to 80 µ or less. Obviously this will never happen in bivalve stomachs. However, I have also noticed a marked difference in average partide diameter in the stomach and intestine of Macoma balthica, but examination of freshly collected individuals showed that, although the majority of partides are less than 50 µ, distinct zones with particles up to 300 µ occur, indicating a rhythmic emptying of the stomach. With regard to the enzymes of crystalline styles it is generally accepted that they play a significant role in extracellular digestion. On .the basis of the work of Christie et al. (1970), an additional function is suggested. The authors found a considerable detachment of Enteromorpha zoospores (attached to cover slips) when treated with ix-amylase at a low concentration (0.01 mg/ml). The ix-amylase of styles may be active in the same way detaching microorganisms on sand grains and detrital partides. In this study a low concentration of style material was found to influence the sinking rate, i. e. sorting of partides. However, it is unknown how viscosity acts in the hydrodynamic situation of an intact bivalve stomach. It should be noted that Hoyt (1970) found the turbulent-flow viscosity of high molecular colloids much reduced, though laminar-flow measurements showed increased viscosity compared with pure sea water. Style solutions have high buffer capacities (Berkeley, 1959), but the pH of style solutions vary in different species. In marine bivalves pH seems·to be slightly acid (5.6-6.6). In Cardium edule I have measured pH of style solutions (demineralized water) to be 6.3-6.4. At this low pH fatty acids formed by lipolysis are not converted into soaps. The occurrence of lipase in bivalve stomachs (Owen, 1966) is in accordance with the presence of an emulsifying property and a stabilized pH caused by crystalline style matter. The advantage of an effective separation of partides with food value, like oil droplets and bacteria, from the structural carbohydrates of detritus is obvious. However, I find the high content of non-enzymatically active protein remarkable. It should be mentioned that proteins e. g. in plasma are known to bind ether compounds and thereby play a role in the absorption and transport of substances. It is, therefore, proposed that these aspects also are considered in future work on crystalline style functions.

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J. HYLLEBERG

KRISTENSEN

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