Holeuryhalinity and its mechanisms in a cirriped crustacean, Balanus improvisus

Comp. B:ochem, Physiol.. 1976~ Vol, 53A, pp. 19 to 30 Pergamon Press Prmlcd m Great arffam

HOLEURYHALINITY AND ITS MECHANISMS IN A CIRRIPED CRUSTACEAN, BALANUS IMPROVISUS H. ]'. FYHN* Duke University Marine Laboratory, Beaufort, NC, U.SA.

(Received 16 December 1974) Almtraet--l. The barnacle Balanus improwsus succeeds in fresh water as well as m sea water. The euryhalinity depends partly on hyperosmotic regulauon of the haemolymph and partly on cell volume regulation. 2. The ammals osmoconform in waters ~ibove 500 mOsm and osmoregulate in more dilute sea waters, showing strict homoiosmoticlty below 100 mOsm. Haemolymph and maxillary gland fluid are isosmottc and have equal chloride concentrations. 3. Seventeen free amino acids are found m thorax muscle tissue in amounts varying with haemolymph osmolahty. Proline reaches unique values of 0 7 M at hypersahne sea water 4 The relative water content of muscle tissue varies httle with changes ,n haemolymph osmolality pointing to a regulation of cell volume. The adjustments of the intracellular amino acids, especially proline, assist in this regulation. 5. The mtermoult cycle does not significantly influence the measured parameters.

INTRODUCTION

amphitrite and B. glandula may be a special feature

CIRRIPEDS make up a quantitatively important component of intertidal and estuarine commumties, thus demonstrating high tolerance to variations in environmental conditions. In 1854 Darwin made a note of the euryhalinity of some Balanomorph clrripeds He found large specimens of the acorn barnacle Balanus improvisus attached to rocks in a small running stream of fresh water in the estuary of La Plata, and observed that the barnacles continued normal cirri beating for many hours when placed in "perfectly fresh water". Although Clrripedla m general is considered to be a marine group, reports are found m the literature of barnacles inhabiting apparently fresh water biotopes as with B. amphitrite (Shatoury, 1958) and B. improvisus (Zullo et al., 1972). The mechanisms underlying the euryhalinity m clrripeds are poorly known and few studies have committed themselves to this problem. Belyaev (1949) found the acorn barnacles B. Balanus, B. balanoides, B. crenatus, and B. improoisus to regulate hyperosmotically. Newman (1967) found B. amphitrite and B. glandula likewise to regulate hyperosmotically, but, contrary to Belyaev he found B improvisus to be an osmoconformer. Foster (1970) concluded that the barnacles B. balanus,

of these species. However, criticism has been raised (Foster, 1970) that the data of Belyaev (1949) and Newman (1967) may not represent animals in steady state, thus, throwing doubt on the osmoregulatory capacity of any barnacle. Belyaev & Newman used less than 3 days of osmotic acclimation in their experiments and Foster (1970) showed that littoral barnacles of the genera Balanus and Elminus needed, on a mean basis, from 5 to 7 days of acclimation to be osmotically equilibrated when transferred directly from 100 to 50% sea water. Individual variation, especially depending upon the level of activity, could more than double the necessary acclimation time in Forster's experiments. This stresses the necessity of using ample acclimation time in studies of the osmotic response in acorn barnacles. This paper presents data on the osmotic response of body fluids and muscle tissue of an acorn barnacle, B. improvisus, in the osmotic steady state condition. The work aims at evaluating the mechanisms of euryhalinity in cirripeds.

B. balanoides, B. crenatus, B. hamert, B. improvisus, Chthamalus stellatus, and Elminuis modestus are osmo-

MATERIALS AND METHODS

Materials The acorn barnacle Balanus improvisus were collected m September 1972 and April 1973 at Cherry Branch, Neuse River, North Carolina, in water of 18-25°C and sahnitles of 2-12%o. The animals were attached to roots of dead cypress trees in the lowest part of the intertidal zone. Pieces of wood each having 100--200 intact animals were brought to the laboratory and kept in aerated aquaria at 23 + I*C Adult animals of 15-20ram basal diameter were used. In the laboratory the animals were exposed to slowly changing sea water osmolality by dripping distilled water or normal sea water (35y00) to the aquaria at a rate which halved or doubled the osmolality in 48 hr. Hypersaline sea water was prepared by adding Rila Sea Salt (Rila Prods., Teaneek, N.J.) to sea water. The animals

conformers. The hyperosmolality found in some instances was ascribed by Foster to the animals not being in a steady state condition. The gooseneck barnacle Pollipes polymerus Is osmoconforming (Fyhn et al., 1972). Thus, conflicting results are found in the literature with regard to the osmotm response of some barnacles i.e.B. Balanus, B. balanoides, B. crenatus, and B. improvisus. The osmoregulation found in B. * Present address: Institute of Zoophysiology, Umversity of Oslo, BImdern, Oslo 3, Norway. 19

20

H.J.

behaved normally throughout the acclimstion period as tions. The dissection, weighing, and freezing of each thorax judged by their clrd beating, feeding and moulting activity took 5-6 rain. Weighings of the thorax exoskeleton from and did not show an evasive dosing up reaction. The ani- exuviae showed that the exoskeleton part of excised thormals were ma~ntaln~ at ~their test osmolalities for 5-10 . axes constitute about 1~ of the thorax wet weight. No weeks before expernnentation to'assure complete acelima- correction his been applied to take account of this. finn. The animals were, fed newly .~tched Artemia nauplii (IVletaframe, San Frafic/sco Bay Brand) every 2nd day and the water was changed weekly. No food was given during Analytical methods the last 1~--2 days before sampling. Animals were mainSea water osmolality was determined with an Advanced rained in sea water of 7 mOsm and 1014 mOsm for more Instruments Osmometer (model 65 31LAS) with an accuthan 8 months without showing signs of growth stagnation. racy of ~ l mOsm. Osmolality of body fluids was determined with a micro-osmometer (Clifton Technical NanoSampling liter Biological Cryostat) with an accuracy of :1:10mOsm. Animals were carefully removed from the wood without The body fluid samples were never exposed to the atmosdamage to the calcareous basement membrane. Mantle pbere. A sample holder with 12 holes was used and samcavity water (5-10 pl) was sampled by forcing the opereular ples of 0'3-1.5 nl were analysed in duplicate or triplicate. plates apart with micro forceps and introducing a glass The sample size did not inituence the osmolality determicro pipette through the aperture. The mantle cavity was minatinn. No change in osmolality was found in samples emptied by another glass mi~o pipette. The animal was left at loading temperature (-0.1°C) for up to l l h r . next mounted upside down in paraffin oil under a low Between each sample the quartz loading pipette was power n~croseope and the calcareous basement membrane washed three times with crom-sulfuric acid, three times and mantle were dissected away. Arty fluid that drained with ethanol, and three times with distilled water. The out was sucked away. Samples (2-5 pl) of maxillary gland pipette was filled and emptied twice with a new sample fluid were taken by microptmcture of the. end-sac/bladder solution before actual loading took place. To emphasize complex located subcutaneously on both sides of the pro- any differences between the haemolymph, maxillary gland soma at the level of the oral cone (Nllsson-Cantell, 1921). fluid, and mantle cavity water, the three fluids from one Samples (5-20 p]) of haemolymph were taken by micro- animal, plus an appropriate standard and distilled water, puncture of the rostral sinus. The body fluid samples were were analysed in parallel. A heat filter was placed in the isolated between paratfm oil in the pipette capillaries and illumination path. The same standard solutions were used immediately frozen at -20"C for later analyses. Sampling with both osmometers. Chloride concentration was determined with a Buchtertook a total of 15--20rain. Micro pipettes were prepared from all*all-free, non-corrosive glass capdlary tubes Cotlove Chloridometer. Sea water samples were read in triplicate. Haemolymph or maxillary gland fluid from 2 (Fischer Sc,ientific Co., No. 5-962-43). Muscle samples were obtained from the thorax. Animals to 5 animals in the same intermoult stage was pooled to were mounted upside down under a low power micro- give adequate quantities (2-5/d) and the chloride conscope, the basement membrane was removed and thorax centration determined from single measurements. Protein concentration was determined according to the dissected free from cirri, oral cone, and hindgut and then cut off and lightly blotted with filter paper. In adult B. method of Lowry (Lowry et al., 1951) using the modificaimprovLms an 'almost pure muscle sample of 2-6 nag wet tion for micro-quantities of Rutter (1967). The samples weight were obtained The excised thoraxes were weighed were read in duplicate at 750 nm on a Cary 141:JVRecordand immediately frozen at -20°C for later analysis, or in~ Spectrophotometer. Crystaliine bovine serum albumen they were incubated at I05"C for dry weight determine- (E]~, ffi 6.6 at 280rim) were used as standard.

1000

E 500 m 0

E

o

E

~.I00

(S)

E

(S)

0

5

!0

50 100 Sea water osmolallty, mOsm

500

1000

Fig. 1. Haemolymph osmolality of intereodyBis Balara~ improvisus acclimated to various sea water osmolalities. The data are given-as mean 4- S.E. with number of anlma]s/11 parentheses.

Euryhalinity in Balan~ lmprovisus The relative water content of muscle tissue was calculated from wet weight and dry weight determinations of single thoraxes on a Sartorius micro-balance (Model 1802) having an accuracy of I/~g. The concentration of cx-amino compounds of muscle tissue was determined on about 10 nag of tissue obtained by pooling thoraxes from 2 to 4 animals in the same intermoult stage. Homogenization (four times) and protein denaturation were carried out in ice-cokl 80~0 ethanol. The precipitate was centrifuged down at 40,000 x g for 15 rain at 4°C in a Sorvall RC2-B centrifuge.The supernatant was evaporated to dryness at 70--750C and the deposit redissolved in 250-700 #l of a sample diluent (citrate buffer (0.2N) of pH = 2-2 with 0.5 mM norleucine) for analyses of ninhydrin positive substances fNPS) and amino acids. The NPS were determined according to the method of Moore & Stein (1948) and triplicate samples were read in a Cary 14u.v. Recording Spectrophotometer at ,570 nm

21

with the sample diluent as blank, Norleucine standards were run with each analysis and the NPS are expressed in m-mole norleucine equivalents per kg tissue water. Amino acid analyses were carried out on a Beckman amino acid analyzer (Model 116) with ten times increased sensitivity. The norleucine of the sample diluent functioned as an internal standard and corrections were applied when the norleucine peak deviated more than 5 ~ from the mean value of the standard runs (19~, of the chromatograms). Blank runs of 80~ ethanol and sample diluent showed ammonia to be present besides norleueine and a correction of 15-6 nmole ammonia/ml sample was applied. Utmost care and cleanliness were emphasized during the analytical work. All glassware were washed in crom-sulfuric acid and thoroughly rinsed in distilled water immediately before use. The ammals were moult staged according to the criteria of Davis etal. (1973).

Table 1. Osmolality of haemolymph, maxillary gland fluid, and mantle cavity water of Balanus improvlsus acclimated to various sea water (SW) osmolalities. (The data are given in m-osmolal as mean 5: S.D. with number of animals in parentheses, or as individual values) Moult stage

Haemolymph

Inter. Pro. Post. All stages

103 + 18(8) 88, 106 94, 103 I0! ___15(12)

Inter. Pro. Post. All stages

104 __.14(6) 109 ___25(4) -106 5:18 (10)

Inter. Pro Post. All stages

124 _+ 9(5) 117 5: 11(4) 110 5:11 (4) 118 5:11 (13)

Inter. Pro. Post. All stages

157 158 141 153

Inter. Pro. Post. All stages

308 + 18 (8) 299, 330 298, 311 309 5:16 (12)

Inter. Pro. Post. All stages

514 515 514 514

+ 19(6) + 15(7) -I- 22(5) + 19(18)

5: 15(4) 5:3 (3) __ 10(3) 5: 10{10)

Inter. Pro. Post. All stages

1031 5: 12(5) 1031 1032 1031 -I- 10(7)

Inter. Pro. Post. All stages

1877 + 7(4) 1870 5:8 (4) 1888 5: 16(3) 1878 5: 12(10)

- - No measurements.

Maxillary fluid SW = 7 mOsm 111 -I- 11 (6) 89, 105 99, I06 106 + II (I0) SW = 29 mOsm 89 + 19(8) 122, 135 -100 5: 25(7) SW -- 54 mOsm 123 +_ 5(5) 123 5: 6(3) 108 4- 12(4) 118 + 11 (12) SW = 106 mOsm 153 5: 11{4) 134 5: 24{4) 133 5: 13(3) 141 + 18(11) SW = 256 mOsm 296 -1- 4(4) 286, 287 293, 350 300 -I- 21 (8) SW = 504 mOsm 483 517, 528 503 508 + 19(4) SW = 1014 mOsm 1027 4- 30(5) --1027 5: 30(5) SW = 1870 mOsm -----

Mantle water 40 -I- 21 (7) 24, 34 40, 56 39 __. 18(ll) 29 5: 4(5) 55 + 39(4) -40 +_ 28(9~ 78 __. 19(5) 6 4 + 7(4) 79 5: 19(4) 74 -I- 16(13) 103, 107 105, 111 111 108 5: 4(5) 266 4- 9 (8) 251 267 265 5:9 (10) 498 5: 8(4) 488, 500 492 + 13 (3) 495 5: 9(9) 1001 _+ 20(6) 1000, 1033 1036 1008 + 22(9) 1867 + 6(4) -1,867 5: 6(4)

22

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Fig. 2 Haemolymph chloride concentration of Balanus trnprovisus acclimated to various sea water osmolalitles. Each point represents measurements on a pooled sample of haemolymph from 2 to 5 ammals of the same mtermoult stage. RESULTS

and the mantle cavity water. An isosmotic condition exists between haemolymph and maxillary gland fluid at a given sea water osmolahty. By using animals of all mtermoult stages and with both fluid osmolalities measured, and taking the ratio of the osmolallties of haemolymph and maxillary gland fluid, a value (mean + S.E.) of 1-034 + 0.028 is reached at for amreals in sea waters from 7 to 106 mOsm, and a value of 1.008 + 0.003 for ammals in sea waters from 256 to 1014 mOsm. The mantle cavity water ~s intermediate in osmolality between that of haemolymph and that of sea water but closest to the osmolahty of the sea water

Body fluid analyses The haemolymph of interecdysis B. irnprovisus is hyperosmotic to sea water and relatively more so at the lowest osmolalities (Fig. 1). Below a sea water osmolality of about 100 mOsm there are only minor changes in haemolymph osmolality while above ca. 500mOsm the haemolymph conforms osmotically with the sea water. The haemolymph osmolality does not change significantly during the intermoult cycle at any acclimation osmolality (Table 1). This applies also to the osmolalities of the maxillary gland fired

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Fig. 3. Difference between haemolymph chloride concentration and sea water chloride concentration at various sea water concentrations of Balanus impromsus acclimated to the respective sea waters Each point represents measurements of a pooled sample of haemolymph from 2 to 5 animals of the same intermoult stage.

Euryhalinity in Balanus zmprovisus Table 2. Chloride concentration of haemolymph and maxillary gland fired of Balanus impromsus acclimated to various sea water chloride concentrations. (The data are given m m-eqmvalents per htre as mean _ S D. wtth number of pooled samples xfl parentheses, or as indwldual wdues) Sea water

Haemolymph

Maxillary fluid

3 14

39 + 4(6) 32 + 3 (3) 38 + 2 (4) 45 __+6(7) 120 -t- 7(5) 259 + 15(6) 507 + 15(6) 958 + 18 (5)

36 _ 10(3) 46 36, 47 41 __+2(3) 135 + 19(3) 239, 276 513, 543

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(Table 1). Also, the variance of the osmolalities of the mantle cavity water, especially at the lower osmolalities, is larger than those of the two other body fluids. This may be caused by contamination of the mantle cavity water with hyperosmot~c haemolymph. Such contamination could have taken place during the sampling procedure by rupturing the soft and vascularized ttssue of the opercular membranes. The hyperosmolality of the mantle cavity water, therefore, may be an artifact rather than resulting from an osmotic regulation of this fluid as suggested by Newman (1967). The chloride concentration of the haemolymph (Fig. 2) decreases proportionally with that of the sea water until a sea water concentration of about 40m-equiv/1 with no further reduction below this level. The intermoult cycle does not seem to have any influence on this parameter. The difference between the chloride concentration of haemolymph and of sea water increases progressively in sea water more diluted or concentrated than ca. 40m-equ]v/1 (Fig. 3). Haemolymph chloride ts hypenonic below this level and hypoionic above it. No difference is found between the chloride concentrations of haemolymph and maxillary gland fluid of animals acclimated to sea waters ranging in chloride concentrations from 3 to 992 m-equiv/1 (Table 2]. The intermoult cycle does not seem to influence the chloride concentration of the maxillary gland fluid and the data of Table 2 concern ammals irrespective of intermoult stage.

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7 200 950 900 900

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Fig. 4. Concentration of ninhydrm positive substances (NPS) of thorax muscle tissue of Balanus improoisus acchmated to various sea water osmolalities. Each point represents measurements on a pooled sample of muscle tissue from 2 to 4 ammals of the same intermoult stage. Protein concentration and concentration of ninhydrin positive substances (NPS) of the haemolymph of B. improvisus at different sea water osmolalities is shown in Table 3. The analyses were carried out on pooled samples of haemolymph from 2 to 3 animals irrespective of intermoult stage. There is an increase m protein concentration at low sea water osmolality while the NPS concentratton remains unchanged. Haemolymph protem concentrations of two other balanids are included in the table showing values of about one third of those in B. zmprouisus at a comparable sea water osmolality

Muscle tissue analyses The analyses of NPS and amino acids were carried out on pooled samples of 2-4 thoraxes. The NPS concentration is independent of the sea water osmolality below 100mOsm and increases with increasing sea water osmolality above this level (Fig. 4). Analyses of ammals in different stages of the intermoult cycle

Table 3 Haemolymph protein concentration and concentration of ninhydrin positwe substances(NPS) of three balanids acclimated to various sea water osmolahties. (The data are given as mean + S.E. of 5 pooled samples) Sea water osmolality (mOsm)

•

Protein concentr, (mgml)

NPS eoncentr. (raM)

10.1 -I- 0"7 5"9 ::h 1"1 7.7 _+ 0"5 2"6 + 0.7 2 5 + 0.4

1-4 + 0'2 -1"9 + 0"2 ---

H.J. FYHN

24

Table 4. Major free amino acids of thorax muscle tissue of Balanus impromsus acclimated to various sea water osmolalities. (The analyses are carried out on pooled samples from 2 to 4 animals of the same mtermoult stage. The data are given m m-moles per kg tissue water as mean + S.D. with number of pooled samples in parentheses, or as individual values) Sea water osmolallty, mOsm Amino ncad

Moult stage

7

29

54

106

256

$04

Arg

Inter Pro Post

I 5 ± 0.3(5) 1 9, 1 9 1'8

15 l8 II

21.25 lost 19

37,38 4,7 45

54±0`7(4) 74 39

86± 77 62

Tau

Inter Pro Posl

0,5 ± 0.1 (5) 0"3. 0`3 0,6

0"5 ± 0-1 (3) I0 lost

11±0-2(4) lost 14

23+0.5(5) 24 22

5 4 ± 0.9(4) 64 62

Glu

Inter Pro Post

15±0"2(5) 1.4, I 8 16

I l :L 0-3(3) I I 10

18±0"3(4) lost 20

2 0 ± 0-6(5) 23 21

Pro

Inter Pro Post

0.2 ± 0"2(51 traces, 0.5 0'4

traces(3) 0.5 lost

0-2 ± 0,314) lost O6

Oly

Inter Pro Post

0.9 ± 0`2(5) 0`7, 0`8 I I

0.9 + 0`2{3) 0,9 I3

Ala

Inter Pro Post

12+0-2(5) I 2, ] 6 I5

1 2 + 0`4(3} I7 2'1

I014

1870

7'6 + 14(6) S5 85

5'7:1:0"8 (3) 6 1.6 7 66

1 5 0 ± 16(5) 16 6 139

166±56(6) 20 3 226

2 9 ± 0.7(3) 2 3, 2 5 37

37±0`5(4) 38 35

5 4 -I- 0-6(5) 68 61

64 ± 18(6) 74 56

91±27(3) 69, 9 8 128

0`4 ± 0`4(5) traces traces

4 4 ± 0,8(4) 44 53

184 ± 29{5) 215 [85

1187 ± 288{6) 1344 1412

509 6 ± 9 2 2 ( 3 ) 577, 613 6742

I 5 ± 0.4(4) lost I8

15_+ 0 5 ( 5 ) 23 I3

8 3 ± 0`8(4) 84 86

163 ± 26{5l 16 0 [55

291 ± 4 1 ( 6 ) 28 2 248

1 9 5 ± 13(3) 15 0, 32 4 269

2-3±0-644) lost l 7

24±0`9(5) 23 3l

84±0`5(4) 64 81

10-9± 13{5) 13 I II 9

163 ± 23(6) I39 140

247 ± 28(3) 277. 30`0 30`6

12(4)

Table 5. Minor free ammo acids and ammonia of thorax muscle tissue of Balanus improvtsus acclimated to various sea water osmolahtles (The analyses are carried out on pooled samples from 2 to 4 animals of the same intermoult stage. The data are given in m-moles per kg tissue water as mean + S.D. with number of pooled samples m parentheses, or as individual values) Sea water osmolahty, mOsm Amino amd

Moult stage

7

29

54

106

256

504

1014

1870

Lys

Inter Pro Post

0`2+00(5] 0"3, 0 4 0`2

02 04 0.3

04, 0.5 lost 0"3

0`6,07 0"5 0-7

10 ± 0-1(4) 14 0,9

12_0"3(4) I2 0-8

1"4_+0'3(6) 0"9 I-5

0'6 :[: 0.1 (3) 0`7, 0'7 0.5

HIs

Inter Pro Post

traces (5) *, 0`1 traces

0,2 0`2 0.4

traces (2} lost traces

O'l, 0.2 0`1 traces

traces (4) " 0`2

0-8 + 0`1 (4) 0.8 0-4

0"7 ± 0.6(6) 0.7 0`7

traces (3) 0.4, 0,6 05

Amm

Inter Pro Post

104-1-0(5) 1,2, I 5 IS

36 &8 27

15.31 lost I3

14.17 I3 0-9

16 ± 0-5(4] 1"0 0`8

2-3 +__ I 5(4) 2"8 4"5

45 + 30(6} 0`4 4 I

52± 12(3) 43 48 36

ASp

Inter Pro Post

0.3 ± 0-1 (5) 0-3, 0 4 0.4

* (3) (t I lost

Irnces (4~ lost *

traces (5) traces 0`I

0-4 ± 02 (4} 0-6 traces

0`2 ± 0`2 (5) traces *

0"3 ± 0,2 (6) 03 traces

2 ] ± 08 (3) I 5, I 6 I6

Thr

Inter Pro Post

0.1 ± 0`0{5) 0`1.0`1 0-3

0`2 ± 0,2(3) 0.2 lost

0"2 ± 0"1 {4) lost traces

traces 15) traces *

02 + 0.0{4) 0`3 traces

I 1 ± 0"6(5) 16 I0

I I + [ 4(6) 22" **

*" (3) traces traces

Ser

Inter Pro Post

0.4+0-1(5) 0.3, 0.3 0.5

0"7±0.1(3) 0`8 10-

0`6 :[: 0`2(4) lost 0.5

U6 ± 0`1(5l 0`6 0.3

0`8 :k 02(4} 09 08

43±11(5) 4-2 2-7

37+28(6) 32 3 I**

7 6 ± [4(3)** I 8, 4 9 65

Val

Inter Pro Post

0'2 + 0,0(5) 0"2, 0.3 0"3

0"4 ± 0.0 (3) 0`4 lost

0-4 ± 0`1 (4) lost 0-7

0.4 ± 0`1 (5l 0-3 0`4

I 2 ± 0 2 (3} I1 07

I 4 ± 0.2 (5) I7 I4

I 6 ± 0`8 (6) 25 20

I 8 ± 0.5(3) I 2, 27 I6

Met

Inter Pro Post

Traces (51 *, trac~i traces

* (3) 0'1 lost

0.1 + 0.0(4) lost 02

0`I ± 0.1 (5) traces traces

0"3 ± 0.0(3) 0`2 traces

0,7 ± 0.I (5) 0,5 0,7

06 ± 0"4 (6) I2 09

traces (31 *, 0-5 06

lie

Inter Pro Post

traces (5) traces, 0.1 traces

traces {3) 0,2 lost

traces (4} lost 0 I

0"1 -I- 0 I (5) traces 02

O3 + 0 0 ( 3 ) 0.3 traces

0`4 ± 0`1(5} 0"5 traces

0"6 _+ 0`2(6) 0-5 0"3

0`7±03(3) traces, 0 7 0`6

Lea

Inter Pro Post

0"I -I-0`I {5) traces, 0.2 0,2

traces (3) 02 lost

0 I + 0`0 (4) lost 03

02 ± 02(5) traces 03

05 ± 0,0 (3) 0`6 traces

0-8 ± 0`2 (5) 0`8 0"6

0'8 "+ 03 (6} I I 0-6

I 4 ± 0`6 (3) traces, I 4 I4

Tyr

Inter Pro Post

* (5) *, 0,2 *

* (3) 0,2 lost

* (4) lost traces

traces {5) traces 0"3

traces (3) 0,2 traces

0.9 + 0.2(5) I0 traces

0-6 ± 0-7{6) 23 0,7

* (3) traces, I 4 I I

Phe

Inter Pro Post

* (5) *, traces *

" (3) 0"2 lOSt

* (4l lost traces

traces (5) traces 0`3

traces (3) 0`2 traces

0"6 + 0`1 (5) 0-9 traces

0.2 ± 0"2(6) 1I traces

* (3) *. 1 I t races

* Undetectable amounts.

** Threonme and serine together.

Euryhahnity in Balanus zmpromsus 500

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1000

m Osrn

F:g 5. Concentration of some free amino acids of thorax 'muscle tissue of Balanus improvisus aechmated to various sea water osmolalities. Mean values of :nterecdys~sanimals are given. Hatehed area includes the other amino acids.

95

25

gave overlapping results. Seventeen free amino acids plus ammonia were found in greatly variable amounts (Tables 4 and 5). At sea water osmolalities below ca. 1(30mOsm no one amino acid dominates the amino acid pool and the concentration of individual amino acids is well below 5 m-moles/kg tissue water. Above a sea water osmolality of ca. 100mOsrn the concentration of some amino acids increases. This applies especially to proline but also to glycine, alanine, taurinc, arginine and glutamic acid (Table 4). Other amino acids do not show such a correlation with the sea water osmolality and their concentration remains at values of 1-6 m-moles/kg tissue water even at high sea water osmolalities (Table 5), Depending upon the sea water osmolality, therefore, there are differences in the total amount of amino acids of the muscle tissue as well as in the relative amount of individual amino acids. The dramatic increase in prohne concentration relative to that of other amino acids is presented in Fig. 5. Mean values of animals in interecdysis are shown. From mere traces (0.2-0.4 m-moles/kg tissue water) at sea water osmolahties of less than 100mOsm the proline concentration mcreases to more than 500 m-moles/kg tissue water at a sea water osmolality of 1870 mOsm. This represents an increase of about 2000 times. Other amino acids of the thorax

\ \ \

¢)

• interecdysis . proecdysis x postecdysis

E 90

\

\

t,.

o 85

:d-'.

.... i- . . . . . . . . . . .

:."--t ..... ".. . 2 ...... • I ~ ......

o}

E

............

~ i-

i ........... i J

\.

"".-.

.... "~-qS,"

i•"-' t- 80 ou

\'-,, \

~ 75

! ",.q,

\

,,

i

"%

\

o~

I,-,.

"%

\

\

70

;

\ I

i

I

I

100 Haemotymph

I

I

I

500 osmotatity,

I

I

I

I

I

1000 m Osm

Fig 6. Relative water content o[" thorax muscle tissue of Balanus improvisus at various haemolymph osmolalities. The three stippled lines show the bchaviour of an ideal osmometer and of solutions of NaC1 and glucose as described m the texL Each point represents measurements on one animal.

~6

H.J. F~a~

muscular.~tm'e o_f B., improv,isus.are also concentrated w,ith increasing sea water osmolality from 100 to l~870.mOsm,.,but this is only in the range of a 2-fold to,20-fold increase. Contrary to proline and most other oS.the amino acids taurine, and possibly glycine and ar~nine, show a decreased concentration.at the hi,ghest sea water osmolality. 'The data in Tables 4 and 5 do not reveal any marked influence of the intermoult cycle upon the concentration of arnino acids of the muscle tissue. The majority of the pro- and postecdysis values (88 and 90°/~ respectively) are within two standard deviations of the mean values of interecdysis animals. Moreover, of the pro- and postecdysis amino acid concentrations deviating more than two standard deviations from the mean value of intereedysis animals, there are equally many above this value (8 and 7~., respectively) and below it (4 and 3%, respectively). The relative water content of thorax muscle tissue remains at about 85% when the haemolymph osmolality is below 300mOsm and there is a progressive decrease with increasing haemolymph osmolalities (Fig. 6), The intermoult cycle is not found to have any significant influence on the relative water content of the muscle tissue. Some calculated lines are drawn into Fig. 6. The osmometer-line describes the expected changes in the relative water content of a tissue exposed to changes in extracellular osmolalities when the cells behave as ideal osmometers. This line is calculated by the following formula (Fyhn et al., 1972) based on a tissue with a relative water content of 84.0% at an extracellular osmolality of 308 mOsm (the actual data for muscle tissue and haemolymph of interecdysis B. improvisus at a sea water osmolality of 257 mOsrn): RW~

=

I00 osm~ osmi + [(100/RWI) - 1] osm2

DISCUSSION

lntermoult cycle In the present study the effects of the intermoult cycle on the osmotic response of B. improvisus seem to be modest and the data from pro- and postecdysis animals do not deviate significantly from the mean values of animals in interecdysis. The accelerating and synchronizing effects of exogenously applied eedysterone (Tighe-Ford & Vaile, 1972) suggest a hormonal control of eirripedian intermoult cycle analogues to that in other crustaceans. Thus, cyclic modifications similar to those in other crustaceans (Passano, 1960; Yamaoka & Scheer, 1970) could be expected during the intermoult cycle in clrripeds. If such modifications in B. improvisus are small or of short duration an extensive material would be necessary to obtain significant differences. In studies on juveniles of B. amphitrite and B. improvtsus, Cosflow & Bookhout (1953, 1956) failed to find any correlation between shell growth and the intermoult cycle. Oxygen uptake rate of intact, adult B. amphitrite did not show a correlation with the intermoult cycle (Costlow & Bookhout, 1958). However, Barnes & Barnes (1964) reported cyclic variations in respiration and water content of excised bodies of B. balanoides taken at timed intervals after ecdysis. Only mean values are given in this paper with no indication as to variance or number of measurements and the significance of the differences throughout the intermoult cycle can not be evaluated. Moreover, the use of excised bodies and the procedure of using the same bodies first in the respiratory experiment followed by determination of body water content, may have adversely influenced these data. Further studies should therefore be carried out to elucidate the effects of the intermoult cycle on cirripedian physiology. An appropriate staging method has recently been developed (Davis et al., 1973) and is a necessary prerequisite for such studies.

RWt and RWz are the relative water contents of the tissue at the original (osm 0 and the changed (osm2) Osmotic regulation of the haemolymph Conflicting results can be. found in the literature extracellular osmolality, respectively. With increasing extracellular osmolality a passive outflux of water with regard to the osmotic response of B. improvisus. would shrink an osmometer and result in a decreasing Haemolymph osmolality measurements have implirelative water content as shown by the osmometer- cated the animal to be osmoregulating (Belyaev, 1949) line. The NaCI- and glucose lines describe the changes and osmoconforming (Newman, 1967; Foster, 1970). in relative water content, mg water/100 mg of solu- These contradicting conclusions may result from diftion, of NaCI- and glucose solutions of different ferent experimental approaches and especially the osmolalities, respectively (Data from Handbook of duration of the acclimation period can have a decisive Chemistry and Physics, CRC, 1972). Tlae lines have influence on the results (Foster, 1970). Evasive behabeen placed within the range of the relative water viour of closing the opercular valves for long periods content of the muscle tissue of animals at low sea of time is typical of acorn barnacles when encounterwater osmolalities by a downward parallel shift. As ing adverse environmental conditions (Barnes et al., the osmolality of the solutions is increased their rela- 1963; Foster & Nott, 1969). When exposed to changes tive water content will necessarily decrease because in sea water salinity the effect of the closing up reacof the admixture of increasing amounts of solute. The tion is to retard the osmotic equilibration between effect is accentuated by increasing molecular weight haemolymph and sea water. Thus, barnacles of the of the solute. Thus, a glucose solution (M = 180.2) genera Balanus and Elminius needed up to 7 days to has a more pronounced decrease in relative water reach a new osmotic steady state when transfexred content on a given osmolality increase than a NaCI directly from 100 to 50% sea water (Foster, 1970). solution (M = 58.5). The actual measurements show In the present study ample acclimation time (5--10 that the muscle fibers are not behaving according to weeks) were used and the data presented describe the an osmometer function but rather that the decrease steady state condition of the animals at the various in relative water content at high sea water osmolali- sea water osmolalities. B. improvisus, ,therefore, posties can result from increasing amounts of solute in scsses a true hyperosmotic regulation of the haemolymph. The regulatory mechanism works efficiently the muscle, fibre protoplasm.

Euryhalinity in Balanus improuisus in highly diluted sea water keeping an osmotic gradient of tS: 1 across the body surface at a sea water osmolality of 7 mOsm. When the sea water osmolality drops below ca. 100mOsm the osmotic regulation relieves the tissue ceils from extensive osmotic stress by maintaining haemolymph osmolality at a constant level. The euryhalinity of B. improvisus therefore, is not solely dependent upon cellular tolerances as has been assu.med earlier (Newman, 1967; Foster, 1970). These authors found that B. improvisus ceased to be active when exposed to sea water salinities of 1-2~'** and the lower tolerance ILrmt was suggested to be about 3~',~(Foster, 1970). The present findings of cirri beating, feeding, and moulting of B. improvisus for more than 8 months in water of 7 mOsm ((>24~/~) extends the tolerance range well into the fresh water region. The successful survival in waters ranging in osmolality from that of fresh water to that of full strength sea water characterizes B. improvisus as a holeuryhaline species in the terminology of Kinne (1964). The mechanism of the hyperosmotic regulation in B. improoisus is not known. The haemolymph proteins will set up a Donnan equilibrium across the body surface resulting in an unequal distribution of the permeable ions and a hyperosmolality of the haemolymph. The magnitude of these effects would increase with increasing concentration and valence of the proteins, and with decreasing concentration of the permeable ions (Bull, 1971; p. 177). Only at concentrations of the permeable ions below 3(X)-400mM can the proteins cause a significant hyperosmolality (Bull, 1971; p. 176) It was, therefore, thought to be of significance that in B. improoisus hyperosmotic regulation is ewdent only at sea water osmolalities below ca. 500 mOsm (Fig. 1). Should the hyperosmolality of B. improvlsus be caused by a passive Donnan equilibrium it would be expected that the haemolymph protein concentration was high and possibly would increase with decreasing sea water osmolality. In B. improvisus, however, the protein concentration (Fable 3) is only 20-30% of that found in other crustaceans (Prosser, 1973) and there is only a small increase from the osmoconforming to the osmoregulating state. Moreover, the chloride ratio between haemolymph and sea water is not in accord with the predictions from the Donnan equilibrium. The haemolymph pH of B. tmprovisus is not known but may be similar to that of another cirriped, the gooseneck barnacle Polliczpes polymerus, where pH values close to 7 were found in submerged animals (Fyhn et al., 1972; Petersen et al., 1974). At this pH the haemolymph proteins will carry a net negative charge. The chloride ratio of a Donnan equilibrium should be less than unity and should decrease with decreasing sea water chloride concentration. The opposite is found in B. improvisus when regulating strongly hyperosmotically (Fig. 3). At chloride concentrations of the sea water below 40--50m-equiv./1 the chloride ratio is above unity and increases with decreasing sea water concentration. These findings on haernolymph protein concentration and chloride ratio, therefore, does not seem to involve a Donnan equilibrium in the establishment of the hyperosmolality in B. improvisus. The hyperosmotic regulation may be governed by active ion transport mechanisms as generally is found

27

in osmoregulators. Haemolymph chloride concentration and haemotymph osmolality changes in parallel and there is a high chloride concentration of the haernolymph in animals at low sea water osmolality (Fig. 2) as is typical of hyperosmotie regulators depending upon ion transport (Prosser, 1973). In B. improvisus the contribution of chloride salts to the haemolymph osmolality seems to be higher in the osraoconforming state than in the osmoregninting state. The chlorides constitute about 50% of haemolymph osmolality in sea waters above 500 mOsm but only about 3(P/0 in sea waters below 100mOsm (Tables 1 and 2). With cationic counterions, probably mainly sodium, the chloride salts would make up the bulk of the haemolymph osmolality in the osmoconforming state and about 60°/0 of it in the hyperosmotic state. Haemolymph NPS concentration does not increase at low sea water osmolality (Table 3) and other solutes must make up about 40°/0 of the haemolymph osmolality at low sea water osmolalities. Proline could be a solute in question. This amino acid could accumulate in the haemolymph after being liberated from the muscl~ tissue during hyposmotic acclimation (Fig. 5). Proline is poorly detected by the ninhydrin procedure and no analysis of haemolymph proline has been made in B. improvisus. In cirripeds the maxillary glands are retained in adult life (Nilsson-Cantell, 1921). Little attention has been paid to the function of these glands but they are assumed to be analogous to the antermal glands of malacostracans (Robertson, 1960; Riegel, 1971). In B. improvisus maxillary gland fluid and haemolymph are isosmotlc and have the same chloride concentrations even when the animals exhibit strong hyperosmotic regulation (Tables 1 and 2). The maxillary glands, therefore, do not seem to be involved in the osmotic regulation of the haemolymph. In the osmoeonforming gooseneck barnacle P. polymerus fluid isosmotie with haemolymph was found to be secreted in the capitulum and assumed to be an ultmfiItrate of the haemolymph, possibly secreted by the maxillary glands (Fyhn et al., 1972). Cirripeds, therefore, seem to be similar to marine and brackish water decapod crustaceans where urine:haemolymph ratios of unity are found with respect to osmolality and chloride concentration (Robertson, 1960; SchmidtNielsen & Laws, 1963; Sehoffeniels & Gilles, 1970b). In malacostraeans the ability to produce urine that is hyposmotie to haemolymph has been correlated with the presence of a nephridial canal in the antermal gland (Schmidt-Nielsen & Laws, 1963; Kirselmer, 1967). It seems of significance thaf in eirripeds the maxillary gland does not include a secretory tubular part (Nilsson-Cantell, 1921). Above a sea water osmolality of about 500 mOsm the haemolymph of B. improuisus conforms osmotically with the sea water but is' slightly hyperosmotic to it (Table 1). This agrees with findings in other barnaeles (Belyaev, 1949; Newman, 1967; Foster, t970; Fyhn et al., 1972) and seems to be a general feature of cirripeds. No explanation has been offered for this observation. However, in the gooseneck barnacle P. polymerus the haemolymph was found to be under high hydrostatic pressure (Fyhn et al., 1973) and it was calculated that the hyperosmolality of the ha~molymph could be balanced by this hydrostatic pressure

28

H.J." Fvn~

levels (106m-moles/kg wet weight) have been found m the lobster Homarus oulgaris (Camien et al., 1951). In the barnacle B. balanoides a seasonal variation from 5 to 35pmoles/100mg dry weight is found in the proline concentration of the body (Cook et aL, 1972). This variation, the authors assumed, reflects food availabihty and reproductive activity of the animals. However, it may also result from seasonal fluctuations in sea water salinity since this parameter was not controlled by Cook et al. (1972). Also, the proline Cell volume regulation concentration of muscle tissue of B. balanoides may Adjustments of the intraeellular concentration of be expected to be higher than the value of 35 pmoles/ ,,-amino compounds under conditions of varying 100 nag dry weight of the body since crustacean musextracellular osmolality has been termed the isosmo- cle tissue generally has higher proline concentration tic intracellular regulation (Jeuniaux et al., 1961). The than other tissues of the body (Schoffeniels & Gilles, present data show that B. improvisus partly depends 1970a~ High proline concentration, therefore, may be on this regulation for its euryhalinity thus including a characteristxc feature of barnacle muscle tissue at cirripeds with the many other groups of marine inver- high sea water osmolalities. In hypersalme sea water proline dominates the intebrates which rely on this mechanism (Schoffeniels & Gilles, 1970b). On a decrease of the extraceUular tracellular arrant acid pool of B. lmprooisus and conosmolality a reduction in the amount of intracellular statutes 86~ of the total concentration of amino acids osmotically active substances will counteract the (Tables 4 and 5; Fyhn, 1974) In other non-eirripedian osmotic inflow of water thereby stabilizing the cell crustaceans prolme make up from 5 to 30~ of the volume (Lange & Fugelli, 1965). In B. improvisus the total amino acid concentration of muscle tissue relative water content of the thorax musculature (Schoffenlels & Gilles, 1970a), while in the barnacle varies less with changes in haemolymph osmolality B. balanoides proline levels up to 53~ of the total than would be expected from a passive osmometer amino acid concentration of the body has been found behaviour of the muscle fibers (Fig. 5). This regulation (Cook et al., 1972). Moreover, the variation in proline of the cellular hydration implies a regulation of cell concentration of about 2000 times found in B. improvolume since water is the major component of the oisus when acclimated from low to high sea water cells. The recorded decrease in the relative water con- osmolality (Table 4), is greater by a factor of 10 than tent at high haemolymph osmolality, most likely, Is what is found in other crustaceans (Schoffeniels & a secondary effect of increasing concentrations of in- Gilles, 1970b). The present findings, therefore, show tracellular solutes and does not indicate a decrease an unusually high utdlzation of proline in the isosmoin cell volume. This is supported by the likeness of tic intracellular regulation of B. improvisus and point the decrease m the relatwe water contents of the to proline metabohsm as a factor of major impormuscle tissue and ofsolutions of NaCI and glucose un- tance for the euryhalinity of this anLrnal. A strong der conditions of increasing osmolalities. The de- and selective adjustment of prohne during hyposmocrease in relative water content of the solutions is tic acclimation would explain the finding in the goosean apparent one being a passive consequence of re- neck barnacle P. polymerus of a cell volume regulaplacement of solute for water and of increase in speci- tion apparently without the participation of or-amino fic weight of the solution. Lange (1964) pointed out compounds, measured as NPS, (Fyhn et al., 1972) that a linear correlation exists between the relative since changes in proline concentration would not be water content of a tissue and the extracellular osmola- detected by the nlnhydrin procedure. lity, and suggested that the coefficient of the correlaHow the accumulation of proline at high haemotion was dependent on the mean molecular weight lymph osmolality is brought about is at present unof the osmotically active substances. This should espe- known. In decapod crustaceans Schoffemels & Gilles cially apply to the substances being exchanged across (see reviews by Schoffeniels & Gilles, 1970b; Schofthe plasma membrane during the process of cell feniels, 1973) have shown in o~tro that the activity volume regulation (Lange & Mostad, 1967). In the of various enzymes involved in amino acid metabolism case of B. improvisus the data of muscle tissue water is dependent upon the concentration of inorganic ions contents are intermediate between those of a NaCI- thus suggesting a control of the cellular amino acid and a glucose solution suggesting strong participation pool by the inorganic ions of the cytoplasm. The of solutes with molecular weights between those of enzymes directly related to proline metabolism have NaCI and glucose in the cell volume regulation. The not been investigated but the possibility exists that steep increase of amino acid concentration, especially such control is in play for this imino acid as well proline concentration (F~g. 5), with increasing haemo- and that this mechanism is applicable to B. improlymph osmolality seems to display such participation. visus. Dramatic increases in proline concentration correlated with increasing osmolality have been f~und Proline concentration in other biological systems i.e. in plants where increasIn thorax musculature of B. improvisus proline may ing water stress and wilting result in proline accumureach values of 674 m-moles/kg tissue water (Fable lation (Barnett & Naylor, 1966; Routley, 1971; 4). Such concentrations are highly unusual among Hubak & Guerrier, 1972). Exogenous application of crustaceans where proline concentrations of muscle proline increases drought resistance in the grass tissue normally range from 10 to 30m-moles/kg wet Carex setifolia indicating that proline is an aetwe weight (Schoffeniels & Gilles, 1970a) even if higher drought resistance factor and not only a product of

thus bringing the animal into a functional isosmotic state (Fyhn et al., 1972). It is not known whether high internal pressures exist in B. improvisus but the finding of internal peak pressures up to 430 mm Hg upon disturbance of B. nubilus (Tait & Emmons, 1925) and the anatomical arrangement of criss-erossing muscles and fibers in the body and mantle of B. balanoides (Gutman, 1960) seem to be in favour of such a possibility.

Euryhalinity in Balanus improvisus the wilting process (Hubak & Guerrier, 1972): Thus, the increase in proline concentration in B. improvisus, correlated with increasing osmolality, may reflect a general mechanism which controls nitrogen metabolism to augment proline accumulation and thereby increase the cellular tolerance to conditions of high extraeellular osmolality, SUMMARY

Balanus improvisus succeeds in waters ranging in osmolality from that of fresh water to that of hypersaline sea water. The euryhalinity depends partly on hyperosmotic regulation of the haemolymph and partly on cell volume regulation. The animals osmoconform in waters above 500mOsm and osmoregulate in more dilute sea waters, showing strict homoiosmoticity below 100mOsm. Haemolymph proteins and ninhydrin positive substances amount to 7-10mg/ml and 12mM, respectwely Haemolymph and maxillary gland fluid, urine, are isosmotic and have equal chloride concentrations. Sixteen free amino acids plus taurlne and ammonia are identified in thorax muscle tissue. Proline especially, but also glycine, alanine, taurine, arginine, and glutamic acid are concentrated and increasingly so with increasing haemolymph osmolality. Proline concentration increases about 2000 times during the acclimation from fresh water to hypersaline sea water, reaching unique values of 0.6--0.7 M at a sea water osmolality of 1870 mOsm. The relationship between muscle tissue water content and haemolymph osmolallty is not in accord with a passive osmometer behaviour of the cells but, rather, seems to reflect changes in the concentration of intracellular solutes. This points to a regulation of cell volume depending upon adjustments of the intracellular concentration of amino acids, especially proline. The intermoult cycle does not significantly influence the measured parameters. Acknowledgements---The author is indebted to the Director of Duke University Matane Laboratory, Dr. J. D. Costlow for provismn of laboratory facilities. His mterest and encouragement greatly stimulated my efforts. Thanks are also extended to Dr. J. Belling Sullivan for his generosity in providing the use of some equipment needed during the work and to Mr. C. Wflham Davis for identifying the species. This study was supported, in part, by contract NR-104-194 between Duke Umverslty and the Office of Naval Research. The author acknowledges addRmnal support from the Norwegian Research Councd for Science and the Humamties, and from The American-Scandinavian Foundatmn.

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29

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