Some aspects of free amino acid metabolism in developing encysted embroyos of Artemia salania, the brine shirmp

Comp. Biochem. Physiol., 1967, VoL 20, pp. 245 to 261. Pergamon Press Ltd. Printed in Great Britain

SOME ASPECTS OF FREE AMINO ACID METABOI,ISM IN DEVELOPING ENCYSTED EMBRYOS OF ARTEMIA SALINA, T H E BRINE SHRIMP D A V I D N. E M E R S O N * Department of Biological Sciences University of Alaska, College 99735, Alaska, U.S.A. (Received 2 6 J u l y 1966)

A b s t r a c t - - 1 . Free amino acid patterns during the hatching of Artemia depend

upon embryonic development and external salinity. 2. High transaminase activity indicates rapid turnover of alanine, glutamic acid, and aspartic acid. This turnover is probably related to protein reorganization. 3. Alanine patterns before emergence are correlated with patterns of enzyme activity and oxygen consumption. A hydroxypyruvate pathway would explain a rapid fall of alanine before emergence. 4. Amino acids are released into distilled water in greater quantities than the observed loss from Artemia suggesting continuous synthesis and loss of amino acids. The loss depends mainly upon the absence of Na +. 5. Internal free amino acid levels after emergence are higher in increased salinity, but the increase contributes only slightly to internal osmotic pressure. Ammonia excretion rate is less during the buildup of free amino acids in greater salinities. INTRODUCTION WhEN encysted embryos of A r t e r a i a salina are placed in water (hydration) the embryos resume development. After an interval of time depending upon conditions of incubation, excystment takes place in two stages (Whitaker, 1940). T h e first stage (emergence) occurs when the hard outer cyst wall splits, and the embryo emerges head first within a hatching membrane. T h e second stage (hatching) occurs a few hours later when a nauplius larva swims from the membrane and shell. Free amino acids have been studied in most of the invertebrate phyla, especially the molluscs and the arthropods (Allen, 1961; Awapara, 1962; Chen, 1962; Lockwood, 1962; Potts & Parry, 1964) and during developmental stages of many animals (Deuchar, 1962). M u c h of the interest in the free amino acids in invertebrates has been limited to their possible role in osmoregulation. Experimental studies on a variety of molluscs and crustaceans have shown that the concentration of free amino acids increases with increasing salinity of the environment. T h e * Supported in part by NIH predoctoral fellowship (No. 1-FI; Gm-21.084) while the author was at the University of South Dakota. 245

246

DAVID N. EIVI~ON

largest quantitative changes in crustacea occur for glutamic acid, proline, glycine, and in some species, alanine. T h e r e are only two brief reports dealing with free amino acids in Artemfa. Bellini (1960) measured total free amino acids f r o m hydration through to several hours after hatching. T h e free amino acids increased steadily until emergence. After emergence, the increase became m o r e marked. Dutrieu (1960) did a semiquantitative survey of free amino acids in encysted embryos, nauplii, and adults. I n her experiments on developing embryos there was an increase of some free amino acids (proline, alanine, glycine, arginine, and threonine) and a decrease in others (glutamic and aspartic acids) by the time of hatching. Dutrieu suggested that the increase of amino acids could be due to intrinsic causes of embryonic development; to the osmotic effects of the outside environment; or to a combination of these two effects. T h i s paper is a quantitative study of the free amino acid levels of developing Artemia embryos f r o m hydration to several hours after hatching. I t demonstrates that the amino acid levels are a function of embryonic development, and that external conditions of the hatching media significantly modify these levels. Changes in levels of certain free amino acids are correlated with oxygen consumption, transaminase activity, and excretion of nitrogen compounds. MATERIALS AND M E T H O D S The encysted Artemia embryos used in this study were obtained in 1965 from Ward's of California, Monterey. The cysts were from Great Salt Lake, Utah. Analysis offree amino acids. Artemia were incubated in three basic media: distilled water, 0"5 M NaC1, and 1"0 M NaC1. Five replica samples of 100 mg original dry cyst weight were taken from each solution at 4- or 8-hr intervals. The animals were filtered from the incubation media (Whatman No. 1 paper) and washed into glass grinding vessels with 70% ethanol. The samples were homogenized in the 70% ethanol with a Teflon pestle and heated in a boiling water bath to flocculate protein. The homogenate was centrifuged to remove coarse material. The pellet was washed twice with hot 70% ethanol to insure complete extraction of free amino acids. The amino acids in the combined ethanol fractions were separated on Amberlite IR-120 (H+), chromatographed on Whatman No. 1 paper, and quantitated with a densitometer as described by Emerson & Duerr, (1966). Recovery studies were done by treating known amounts of standard amino acids in exactly the same manner. Calculations. All calculations are based on the dry weight of the embryo. Since the weight of the cyst shell is 22 per cent of the total cyst weight (Clegg, 1962), the weight of the embryonic material is 78 per cent of the total cyst weight. Water analysis. All incubation media contained 100 units of penicillin and 100/zg of streptomycin per ml (Clegg, 1964). Exactly 20"0 ml of solution were measured into sterile petri dishes and 100 mg cysts were incubated for various periods of time at 25°C. The water was filtered into clean test tubes, and frozen immediately for later analysis. Aliquots of 2"0 ml were analyzed for ammonia using the microdiffusion method of Seligson & Seligson (1951). Urea was determined by means of the quantity of ammonia liberated by urease (Sigma urease solution in 50% glycerol). Aliquots of 0"5 ml were analyzed for total ninhydrin-positive substances (NPS) by the method of Troll &Cannan (1953) using leucine as a standard. All colorimetric measurements were made on a Bausch & Lomb Spectronic 20 colorimeter. Appropriate blanks and standards were always used. Amino acids in the water were chromatographed as described above.

ASPECTSOF FREEAMINOACIDMETABOLISMIN THE BRINESHRIMP

247

Assay of enzyme activity. Samples of 100 mg dry cyst weight were incubated at 25°C in 0"5 M NaC1 and removed at intervals. The cysts were washed into grinding vessels from filter paper with 10"0 ml of 0.35 M phosphate buffer (pH = 7'5). Homogenates were made in ice-cold buffer and frozen overnight. The thawed preparations were filtered through Whatman No. 1 paper. Aliquots of the crude filtrates were used for enzyme analysis. Transaminases. The spectrophotometric method as outlined in Sigma Technical Bulletin No. 410 was followed except that 0"5 ml of tissue extract was used instead of 0'2 ml serum. All reagents were obtained from Sigma Chemical Co. (St. Louis, Missouri). The transaminases studied were: glutamic-oxalacetic transaminase (GO-T) which catalyzes the reaction, aspartate + a-ketoglutarate = pyruvate + glutamate; and glutamic-pyruvic transaminase (GP-T) which catalyzes the reaction, alanine+ c~-ketoglutarate=pyruvate+ glutamate. The reactions were followed by measuring the rate of decrease of O.D. as fl-DPNH is oxidized at 340 m/z in a Beckman DU spectrophotometer. Cuvette temperature was takenafter eachset ofreadings (30-31°C) and appropriate correction made. Calculations are given in International Units (I.U.) of enzyme activity per mg dry embryo weight. One I.U. is equivalent to the conversion of 0"001/zmole of substrate/min. Glutamic dehydrogenase (GDH). This enzyme catalyzes the reaction, L-glutamate-+ D PN + = ~-ketoglutarate 2- + DPNH + NH4 + + H +. The reaction was followed by measuring the rate of disappearance of/~-DPNH at 340 m/z in the presence of ct-ketoglutarate and NH4C1 using a modification of the method of Olson & Anfinsen (1952). Each cuvette contained 0"2 ml/~-DPNH (1 mg/ml) ; 0"5 ml 1"0 M NH4C1 (in 0"35 M phosphate buffer, pH 7"5); 1"0 ml extract; and 1"1 ml water. After 10 min, 0"2 ml ~-ketoglutarate (0-1 M) was added, and the decrease of OD340 was followed for 6 min. Osmotic pressure values. Osmotic pressures of sucrose solutions were estimated from data given by Garner (1928). The formula 7r=(C x G)RT was used to calculate osmotic pressure of salt solutions, where r: is the osmotic pressure in atmospheres; C, the molarity; (;, the cryoscopic coefficient; R, the gas constant; and T, the absolute temperature. Values for G are from Heilbrunn (1956). Internal osmotic pressure in Artemia due to free amino acids was estimated with the above formula without using the G factor. In these calculations, the water content of the embryos was assumed to be 75 per cent (Dutrieu, 1960). RESULTS

Free amino acid levels during development T h e changes of free amino acids during development in 3 salinities are summarized in Tables 1, 2, and 3. T h e overall patterns of free amino acid changes are shown in Fig. 1. T h e times at which the first emerged embryos appear are 12 hr in distilled water, 16 hr in 0.5 M NaCI, and 24 hr in 1.0 M NaC1. T h e time required for 50 per cent of the viable embryos to emerge (TsoE) was estimated from counts of emerged nauplii. TsoE in distilled water is 20 hr; 24 hr in 0.5 M NaC1; and 44 hr in 1.0 M NaC1. All amino acid values are corrected for recovery. Free amino acid levels as a function of embryonic development. T h e levels of free amino acids appear to be a function of both embryonic development and external salinity. Changes due to embryonic development can be most clearly seen during the first few hours after hydration. I n all salinities, alanine, glycine, threonine, histidine, and lysine increase. T h e r e is an immediate decrease of aspartic acid, serine, and arginine. T h e immediate response of the other amino acids is not certain on the basis of the present data. T h e two most notable changes due to embryonic development are the rapid rise and fall of alanine, and the initial decline of aspartic acid before emergence.

248

DAVID N . EMERSON

TABLE 1--INTERNAL AMINO ACID LEVELS OF Artemia INCUBATED IN DISTILLED WATER AT

25 °C Hours of development Alanine Glutamic acid Proline Aspartic acid Serine Glycine Threonine Tyrosine Arginine Histidine Phenylalanine Lysine Cystine Valine Leucine-isoleucine Total

0

4

8

12

16

20

24

32

40

1"00 1"42 0"43 1"66 0'42 0'44 0"14 0"62 0"45 0"13 0"01 0"19 0'12 0"15 0"10 7"28

2"34 1"43 0'61 0"69 0"34 0"65 0'37 0'66 0"40 0"21 0"18 0"26 0'12 9'19 0"15 8"60

2"84 1-42 0"80 0'45 0"34 0"47 0"37 0"73 0"39 0'19 0'20 0"29 0"09 0-24 0.19 9.01

2"33 0"45 0"60 0"73 0"56 0"52 0'49 0"59 0"42 0"21 0"15 0"27 0"07 0"14 0"14 7'67

0"78 0.41 0.60 0-35 0"48 0"42 0-39 0.54 0'39 0-01 0'07 0-24 0"02 0"13 0"07 4"90

0"66 0"36 0-31 0"11 0"39 0'32 0"18 0-43 0"33 0"01 0"01 0"18 0"02 0"01 0"02 3"34

0.40 0"27 0"28 0"19 0"08 0"13 0"13 0"24 0"33 0"01 0"01 0-02 0"01 0"01 0"02 2"13

0"40 0" 10 0"21 0"25 0"29 0'16 0"14 0"36 0"33 0"01 0'01 0"02 0"01 0"01 0-02 2"32

0"40 0"10 0"21 0"33 0"23 0"13 0"16 0"34 0"33 0'01 0"01 0"02 0'01 0"01 0"02 2"31

All figures are ~g of amino acids/mg of dry embryo weight. Each value is the mean of 5 replica determinations.

TABLE X--INTERNAL AMINO ACID LEVELS OF Artemia INCUBATEDIN 0"5 M NaCl AT 25°C Hours of development Alanine Glutamic acid Proline Aspartic acid Serine Glycine Threonine Tyrosine Arginine Histidine Phenylalanine Lysine Cystine Valine Leucine-isoleucine Total

0

4

1"00 1 " 9 6 1"42 1 " 2 3 0"43 0 " 1 3 1"66 0"68 0"42 0-37 0"44 0"72 0"14 0 " 3 3 0"62 0"22 0-45 0-40 0'13 0"19 0"01 0 " 1 8 0"19 0'20 0.12 0 " 1 5 0"15 0"16 0'10 0 " 1 3 7.28 7 " 0 5

8

12

16

20

24

32

40

3"32 1.51 0.55 0"61 0-52 0"74 0.53 0"60 0"39 0"19 0"19 0"21 0"18 0'25 0"19 9.98

3-72 1"77 0-64 0"59 0"57 0"68 0-65 0"72 0"36 0-26 0"24 0"32 0"31 0"33 0"23 11"39

3"55 2"01 0"95 0"73 0"75 0"95 0"75 0"82 0"38 0-33 0"26 0"36 0"39 0"37 0"24 12-84

1"91 2-02 1-21 0"76 0"84 1"21 0"78 0"84 0"37 0"37 0"23 0"34 0-38 0"34 0-20 11"80

2'02 2"00 1"25 0"63 0"58 1-31 0"83 0"92 0"47 0"44 0"27 0"39 0"39 0"33 0"20 12"03

2.17 1"75 0-99 0"76 0"89 1"08 0-85 0"80 0"58 0"48 0"22 0'41 0"30 0-27 0"19 11.74

2"30 1"34 1"06 0-65 1"16 0:90 0"87 0"90 0"71 0"51 0"24 0"39 0"36 0"33 0'22 11"94

All figures are/zg of amino acids/mg of dry embryo weight. Each value is the mean of 5 replica determinations.

ASPECTS OF FREE AMINO ACID METABOLISM IN THE BRINE SHRIMP TABLE 3--INTERNAL

Hours of development

AMINO ACID LEVELS OF

249

Artemia INCUBATED IN 1 " 0 M N a C 1 AT 2 5 ° C

0

4

8

12

16

20

24

28

32

36

44

52

Alanine Glutamic acid Proline Aspartic acid Serine Glycine Threonine Tyrosine Arginine Histidine Phenylalanine Lysine Cystine Valine Leucine-isoleucine

1.00 1"42 0"43 1.66 0.42 0-44 0-14 0-62 0'45 0.13 0.0l 0.19 0.12 0.15 0"10

1"45 1'55 0'36 0'78 0"28 0"46 0"27 0'43 0"43 0-18 0.01 0.26 0.14 0-11 0"08

2"06 1"18 0.48 0"55 0'33 0"48 0.24 0"53 0'37 0.14 0"01 0"24 0"17 0"16 0-15

1.79 1.18 0'52 0"69 0.41 0'61 0"30 0"58 0-45 0"20 0"01 0.26 0'21 0"15 0"11

1.13 1.01 0.48 0.53 0'37 0.48 0-35 0-67 0'37 0.14 0.01 0.23 0.26 0"19 0"14

1.83 0.86 0.47 0.63 0.56 0.70 0-40 0.69 0.46 0.23 0.01 0.32 0.30 0-19 0-13

1.85 1.00 0.50 0.75 0.47 0"73 0.40 0"77 0.48 0.29 0.23 0.34 0.33 0"21 0"14

2"29 1-36 0.90 0"83 0"72 0'79 0"70 0"88 0.40 0"24 0"20 0"30 0'32 0"26 0"15

2-80 0.69 1.31 0.89 0.96 1.13 1.13 0.79 0.54 0.34 0.22 0.37 0"44 0.24 0"19

2.89 0"90 1.43 1.14 1.25 1.28 1.30 0.85 0'55 0.28 0.22 0.35 0.44 0.31 0"19

2.92 0.88 1.54 1.11 1.25 1"28 1"30 0"94 0.69 0"32 0.24 0"33 0"46 0"33 0"21

2.90 0.83 2-04 1.15 1.27 1.20 1.30 1-01 0.69 0.44 0-32 0.43 0-45 0.39 0'32

Total

7"28 6"79 7"09 7'47 6"36 7"78 8"4910"3412.0413"3813-8014'14

All figures are/zg of amino acids/mg of dry embryo weight. Each value is the mean of 5 replica determinations.

o

T50%E

Ts~°/°E

~20 V

/o . . . . r -

5,. b -~ / o

° Y

/"

0rV~-,--'-:'v.,/ #"..,.._ ./"0. " e .......... • ............•

~ot', 0

~ 8

,

:

~6

, 40

.

24 Development,

32

, 48

hr

FIG. 1. Total free amino acid nitrogen in Artemia during development in 3 salinities. Ts0 % E is indicated by arrows to show comparable times of development. T h e figures of Tables 1, 2 and 3 were converted to amino acid N and totaled for each hour. Incubation in distilled water is shown by the dotted line, in 0"5 M NaCI by the solid line, and in 1-0 M NaC1 by the dashed line.

Free amino acid levels as a function of salinity of the incubation medium. All of the free a m i n o acids decrease to low levels after e m e r g e n c e i n distilled water. C o m p a r i s o n of free a m i n o acid levels after Ts0 % E of Artemia i n c u b a t e d i n 0.5 M

250

DAVID N. EMERSON

and 1.0 M NaC1 show that all of the amino acids, except glutamic acid and histidine, are higher in 1.0 M NaCI than in 0.5 M NaC1. Internal osmotic pressure due to free amino acids after hatching. An external increase of osmotic pressure is accompanied by an internal increase of osmotic pressure due to free amino acids. The internal pressure, however, does not increase proportionally to the external pressure (Fig. 2). E

30

/

o 10

E o

// 0

__,;~

oressure(ornino acids)

0"5

Salinity,

1.0 NaCL

FIG. 2. T h e relationship between external osmotic pressure and internal osmotic pressure in Artemia caused by free amino acids. The internal osmotic pressures were calculated by converting values from Tables 1, 2 and 3 into /zM using averages of 21 10 hr for distilled water and 0"5 M NaC1, and 36-52 hr in 1"0 M NaCl.

Water analysis Urea. Urea was not detected in the water. This is in agreement with Bellini & de Vincentiis (1960a, b). Ammonia. Ammonia excretion into the water begins shortly after hydration. The rates of ammonia excretion are determined by the salinity of the incubation media and by the time of development (Table 4). TABLE 4

HOURLY RATES OF AMMONIA EXCRETION BY Artemia DURING INCUBATION IN 0"5 1~I AND 1"0 M NaC1 AT 25°C

Development period 0"5 M NaC1 1"0 M N a C I

Period of maximum increase of internal free amino acids* 0-16 hr 16-32 hr 0"0134/zg/hr

Period after T60 % E 24--40 hr 44 52 hr 0"0033/zg/hr

0'0077 F g / h r

0"0026 F g / h r

* Determined from Fig. 1. All figures are Fg of ammonia N in the water/rag of dry embryo weight.

Ninhydrin-positive substances (NPS). Analysis of 0.5 M and 1.0 M NaC1 shows low levels of NPS which can all be accounted for as ammonia. Incubation in distilled water, however, results in the release of small amounts of ammonia and

251

ASPECTS O F FREE A M I N O A C I D M E T A B O L I S M I N T H E B R I N E S H R I M P

large a m o u n t s o f N P S into t h e w a t e r ( F i g . 3). T h e t o t a l a m o u n t o f a m i n o a c i d n i t r o g e n f o u n d in t h e w a t e r (1.25 /~g/mg e m b r y o ) is g r e a t e r t h a n t h e a m o u n t of free a m i n o a c i d n i t r o g e n lost f r o m Artemia (0.93 t~g/mg e m b r y o ) in d i s t i l l e d w a t e r t h r o u g h 24 hr. T h e a m i n o a c i d n i t r o g e n in t h e w a t e r can b e a c c o u n t e d for c h r o m a t o g r a p h i c a l l y ( T a b l e 5). S e v e r a l o f t h e i n d i v i d u a l a m i n o a c i d s a p p e a r in t h e w a t e r in g r e a t e r a m o u n t s t h a n t h e o b s e r v e d d e c r e a s e f r o m Artemia. 15

z"

Artemio amino acid N

o

.~

t - ......... o...

r

~ - oa

...".............. . ""*

1-0

mino acid N

3 z 0-5

o Water ammonia N L ~ o . . . . ? . . . . ~ . . . . r . . . . "- . . . . 7. . . . 0

4

8

12

16

Development,

20

-, F "

24

hr

FIG. 3. Relationship between loss of free amino acids and appearance of nitrogen compounds in water when Artemia are incubated in distilled water. Each point for water amino acid N or ammonia N is the mean of 6 determinations. TABLE 5--PAPER

C H R O M A T O G R A P H I C A N A L Y S I S O F A M I N O ACIDS RELEASED B Y

Artemia

THROUGH

2 4 HR OF I N C U B A T I O N IN DISTILLED WATER ( 2 5 ° C )

Amino acid Glutamic acid Alanine Histidine Leucine-isoleucine Glycine Aspartic acid Serine Threonine Tyrosine Valine Praline Total Amino acid N(colorimetric) % Amino acid N accounted for by chromatography

/zg amino acid N in water per mg dry embryo 0"33 0'28 0"14 0' 13 0' 10 0"09 0'07 0-07 0-05 0-03 trace 1"29 1 '25 t

#g amino acid N decrease per mg dry embryo (8-24 hr)* 0.10 0"38 0"04 0"02 0"06 0"06 0'04 0'03 0"03 0'03 0-07 0"86

103 %

* Calculated from Table 1. t F r o m Fig. 3. All values are the means of 6 replica samples. T h e second column of figures compares the observed decrease of internal amino acids of Artemia with the amount appearing in the water.

252

DAVID N. EMERSON

Total N P S found in the incubation medium at T50% E is a function of chemical makeup and osmotic pressure of the medium (Table 5). Increased osmotic pressure of sucrose causes less N P S to be released. I f NaC1 is added to the sucrose, the N P S released is the same as the NaC1 controls. T h e same observation is true for CaClg, i.e., addition of NaC1 decreases N P S in the water. Other sodium salts (Na~SO4, NaNO3) and salts without sodium (CaCl~, LiC1) also allow more N P S to be released into the water than the NaC1 controls. TABLE 6--TOTAL

N I N H Y D R I N - P O S I T I V E SUBSTANCES SEVERAL SOLUTIONS AT

RELEASED BY

Artemia INCUBATED

IN

T5o%E

Solution

Total NPS*

Distilled water (0) LiCI (16"9) Sucrose (9.2) NaNO3 (16'9) CaC12 (16.9) CaClz (7"7) + NaC1 (9"2) NazSO4 (16-9) Sucrose (16-9) 0'50 M NaCI (16"9) Sucrose (7"7)+ 0"25 M NaC1 (9"2) 0"25 M NaC1 (9"2)

0"885 0'302 0"286 0"182 0"161 0"135 0"130 0"120 0"085 0"078 0"073

* Not corrected for ammonia. All figures are/zg of amino acid nitrogen/rag of dry embryo. Each value is the mean of 5 replica determinations. The numbers in parenthesis give the osmotic pressure in arm.

Enzyme activity during development T h e activities of G P - T and G O - T during development are summarized in Table 7. T h e activity of G P - T closely follows the pattern of alanine changes (Fig. 4). Both transaminases can convert much more substrate than actually is observed to change (Table 8). In addition to the transaminase activity, glutamic dehydrogenase ( G D H ) activity is also present (Table 9). T h e activities of G P - T , G O - T , and G D H at Ts0 % E in the three incubation media are not significantly different ( T a b l e 9). T A B L E 7 - - T R A N S A M I N A S E ACTIVITY DURING THE DEVELOPMENT OF

Artemia INCUBATED

IN

0-5 M NaC1 Hours of development GO-T GP-T

0

4

8

12

16

16"7 17"8 2 0 " 7 17"3 22'8 1.9 2"2 2"3 2"8 3"4

20

24

32

40

19"7 2 5 " 0 23"6 25 7 2.8 2"6 2"8 3"2

All figures are International Units/mg of dry embryo weight. Each value is the mean of 5 replica determinations.

ASPECTS OF FREE A M I N O ACID M E T A B O L I S M I N THE B R I N E S H R I M P

z ~,.¢I b9 " ~

z

8

6~ 66666666

Z

0

66666666

<

0

~'~ ~.~ < z

66666~66 z

<

~.~

'~

0 Z

o

r~

o .~[--

~',~

e~ o oo ,.4

o a"~ g

253

254

DAVID N. EMERSON o

~ /

/

dP

i L\", , ° * . . ...... I " , $~............

I....'/"i\

.

h,a~',ne~._...-o

~,~ o . o 20 o,,, ~

Oxygen

o.o$ I

/. 8~ c,lo 3 ::k'~ o "6

I 8

t

: 16

I 24

Development,

I 32

I

I 40

hr

FIG. 4. Relationship between glutamic-pyruvic transaminase activity, alanine levels, and oxygen consumption for Artemia incubated in 0.5 M NaC1. The oxygen curve is redrawn from Emerson, 1966. The time of appearance of the first emerged embryos is indicated by the vertical dashed line. Ts0 %E is indicated by arrows. TABLE 9--COMPARISON OF ENZYME ACTIVITY AT TS0 % E oF Artemia INCUBATED IN 3 SALINITIES

Enzyme

Distilled water

0"5 M NaCI

1"0 M NaC1

GP-T GO-T GDH

0"018 + 0"002 0"163 + 0"032 0"004 + 0"001

0"019 + 0"002 0"164 + 0"0t9 0.005 + 0"002

0"018 + 0"001 0"170 + 0"015 0'003 + 0'000

All values are decreased in ODaa0 per min per 10 mg dry embryo weight. Each value is the mean of 6 determinations. The numbers preceded by + give confidence intervals at the 95% level. DISCUSSION T h e overall pattern of free amino acid changes during development (Fig. 1) of encysted Artemia embryos depends upon intrinsic embryonic development and conditions of the external environment. Many of the amino acids show a c o m m o n pattern for the first few hours after hydration which can be attributed to embryonic development. T h i s pattern is soon modified by the external salinity. T h e modifications caused by external salinity overlap the embryonic changes of amino acids before emergence, and make it difficult to distinguish between the two phenomena. Embryonic development in some fish and amphibians involves a general decrease of free amino acids which were at relatively high levels during early stages (Deuchar, 1962). In Artemia, however, there is a general increase of amino acids up to T5o% E except when they are incubated in distilled water. Comparison of amino acid patterns for Artemia incubated in different salinities suggests that the patterns are probably a response to external salinity. Bellini's (1960) curve for free N P S in Artemia incubated in 3 % NaC1 (approximately 0.5 M) resembles the pattern of

ASPECTS OF FREE A M I N O ACID METABOLISM I N TIIE BRINE S H R I M P

255

free amino acid nitrogen (Fig. I) in 0.5 M NaCI more closely than it resembles the curves for the other salinities. Likewise, Dutrieu's (1960) curve for non-protein nitrogen in Artemia incubated in sea water plus 1"5% NaCI (approximately 1.0 M) resembles the curve for free amino acid nitrogen in 1.0 M NaCI (Fig. 1). Free amino acid levels as a function of embryonic development The period of development between hydration and emergence is one in which considerable differentiation must occur. Definite structures appear in the emerged embryo which were not present in the encysted embryo (Weisz, 1947). This period of time will be referred to as the differentiation period in this discussion. There is no cell division during this period (Nakanishi et aL, 1962; Emerson, 1963) so that differentiation probably involves cell movement and alteration, and degradation of yolk protein for the synthesis of new structural and enzyme protein. Protein turnover is indicated by a rise of certain proteolytic activity (Bellini, 1957) while total protein N changes very little (Urbani, 1959). Degradation of yolk protein into new protein components of embryonic tissue implies that the free amino acid pool is an intermediate. The amino acid pool is not stable, but involves considerable turnover among at least aspartic acid, glutamic acid, and alanine. Rapid turnover is suggested by the observation that transaminase activity for these three amino acids is capable of turning over much more substrate than is actually observed to change (Table 8). Another line of evidence indicating rapid turn-over of aspartic and glutamic acids is that they become highly labeled if Artemia are incubated in an atmosphere containing C14Oz (Clegg, personal communication and 1965). Rapid labeling of aspartic acid occurs in spite of the observation that its level remains relatively low and constant after its initial decline during the first hours after hydration (Tables 2 & 3). It is well known that glutamic acid plays a central role in exchange of amino groups among many amino acids. Several lines of evidence support the idea that glutamic acid also plays such a role in Artemia embryos. (a) The presence of the transaminases and glutamic dehydrogenase (Tables 7-9) provide a pathway for the conversion of s-amino groups to other nitrogen containing compounds. (b) An interconversion between proline and glutamic acid is suggested by the rise in proline that coincides with a fall in glutamic acid for Artemia incubated in 1.0 M NaC1 (Table 3). (c) When incubated in distilled water, all of the internal amino acid levels decrease (Table 1). However, glutamic acid is the principle amino acid which appears in the water (Table 5). The glutamic acid level in the water is greater than the observed loss from Artemia. These observations suggest that other free amino acids are converted to glutamic acid. The absence of proline, and the presence of relatively large amounts of histidine in the water further suggest interconversions of these amino acids with glutamic acid by pathways known to exist in other organisms. Turn-over of the free amino acid pool requires the presence of o~-keto acids for carbon skeletons of amino acids. The carbon skeletons can come from preexisting amino acids, or supplied through carbohydrate metabolism. During the

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differentiation period, large amounts of trehalose are being utilized (Dutrieu, 1960; Clegg, 1964). Much of the trehalose is used to synthesize glycogen and glycerol (Clegg, 1964), while the remainder as glucose could enter known pathways of metabolism to provide a supply of ~-keto acids. The presence of the transaminases and glutamic dehydrogenase in Artemia provide the necessary link between metabolism of proteins and carbohydrates. Aspartic acid and alanine, which show the most striking changes during the differentiation period, will be discussed in detail. Aspartic acid. Aspartic acid levels fall rapidly in the first 4--8 hr after hydration in all incubation media (Tables 1-3). Decreases in aspartic acid have been observed during embryonic development of other animals: trout (Rizzoli, 1957); amphibians (Kutsky et al., 1953; Chen, 1956; Deuchar, 1956); and Drosophila (yon der CroneGloor, cited by Chen, 1962). It has been suggested that the decrease of free amino acids which were very abundant at initial stages of development of some of these animals may be due to the synthesis of nucleotides as more nuclei are formed during cleavage, or because of rapid metabolism during the utilization of carbohydrates for respiration (Deuchar, 1962). Although there is no cell division during the differentiation period, it has been suggested that aspartic acid could be utilized in pyrimidine synthesis by known pathways (Clegg, personal communication and 1965). This is indicated by the formation of labeled UMP and CMP, in addition to highly labeled aspartic acid, when Artemia are incubated in C140~. In addition to possible utilization for pyrimidine synthesis, aspartic acid may have its amino group transferred to other amino acids which are increasing at the same time. The a-keto acid skeleton may then be metabolized or used in the synthesis of other amino acids. Alanine. Alanine rises rapidly and reaches a peak a few hours before emergence. The alanine pattern resembles the pattern of glutamic-pyruvic transaminase activity (Fig. 4). Trehalose is being utilized rapidly at this time (Clegg, 1964). Such utilization could supply excess acid necessary for the carbon skeleton of alanine. These observations suggest that the transamination reaction has the following direction in Artemia: glutamate + pyruvate = a-ketoglutarate + alanine. Alanine may represent a significant link between carbohydrate metabolism and synthesis of new amino acids during development. It would be an important source of carbon skeletons arising from pyruvic acid as well as a medium for transfer of amino nitrogen. There are several possible explanations for the rapid fall of alanine which coincides with a rise in oxygen consumption and with the appearance of the first emerged embryos (Fig. 4). First, much differentiation has been completed at this time, so that alanine may be no longer necessary as a medium for synthesis of new amino acids. Pyruvic acid may now be utilized mainly for respiration. A second explanation for the decline of alanine is that trehalose is more or less used up at this time (Dutrieu, 1960) and pyruvic acid may no longer be in excess. The embryo in fact has switched from carbohydrate metabolism to mainly lipid metabolism as evidenced by lowered respiratory quotients (Dutrieu, 1960; Emerson, 1963), an

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increase in lipase activity (Bellini & Lavizzari, 1958), and a decrease in total lipids (Dutrieu, 1960; Urbani, 1959). Pyruvic acid may no longer be in excess for alanine synthesis. A third explanation involves the possibility of a pathway involving hydroxypyruvate. Such a pathway has been proposed for the lobster, Homarus vulgaris L. (Gilles & Schoffeniels, 1964 a, b). On the basis of tracer studies, they suggest that glucose can go directly to the synthesis of dicarboxylic acids by means of a hydroxypyruvate shunt without going through Kreb's cycle. In view of a decrease of glycerol (Clegg, 1962) and alanine (Fig. 4) and a rise of serine (Table 2) at about the same time, the following reactions would explain the observations, assuming a hydroxypyruvate pathway exists. (a) glycerate = hydroxypyruvate (b) hydroxypyruvate + alanine = serine + pyruvate The overall reaction is: glycerate + alanine = serine + pyruvate. Reaction (a) requires DPN, known to be present in Artemia embryos (Warner & Finamore, 1965) and a dehydrogenase. Reaction (b) requires an alanine-pyruvate transaminase. Such a transaminase has not been demonstrated in Artemia, but is known to exist in other animals. The overall reactions shows that glycerol and alanine would decrease at the same time, and could provide a large amount of pyruvic acid as substrate for the respiratory increase (Fig. 4) which occurs at this time. The present study has demonstrated directly or indirectly that the following reactions may occur. (1) NHa + a-ketoglutarate = glutamate (2) Amino acid + a-ketoglutarate = glutamate + o~-keto acid (3) Aspartate + c~-ketoglutarate = oxalacetate + glutamate (4) Glutamate + pyruvate = a-ketoglutarate + alanine Reactions (1) and (2) would provide a pathway for amino acids and amino nitrogen into the free amino acid pool. The reverse ofreaction (2)would provide a mechanism for synthesis of new amino acids. The four reactions can occur singly or coupled in various combinations. The net result is that aspartic acid, glutamic acid, and alanine are central points of transfer for amino nitrogen derived from yolk protein, and for carbon skeletons derived mainly from carbohydrate metabolism. An obligatory role of transaminations in amino acid biosynthesis and degradation is indicated from a number of studies on micro-organisms and animals (Cohen & Sallach, 196]). Transaminations probably have a similar role during the development of Artemia. Free amino acid levels as a function of salinity of the incubation medium Incubation in distilled water. Incubation in distilled water is an abnormal condition which results in a decrease of all free amino acids of Artemia to low levels (Table 1, Fig. 1). Although this decrease becomes especially apparent after 8 hr of development, amino acids appear in the water within 2 or 3 hr after hydration (Fig. 3). The decrease of internal amino acids is probably not due to a decrease

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of enzyme activity involved in amino acid metabolism, because the activities of the enzymes studied do not change for Artemia incubated in different salinifies (Table 9). In fact, the total amino acid nitrogen levels (Fig. 3) and quantities of some individual amino acids (Table 5) which appear in the water are greater than the observed loss of free amino acids from Artemia. This observation suggests that free amino acids are being synthesized but are lost into the water. The loss of amino acids does not seem to depend upon charge. For example, glutamic acid and histidine, which are acidic and basic amino acids respectively, would be expected to have different charges at physiological pH, but both appear in the water at higher levels than the observed loss from Artemia. Development in distilled water proceeds to emergence, but not to normal hatching of motile nauplii. Emergence is probably made possible by water uptake due to increase of internal osmotic pressure caused by glycerol (Clegg, 1964) and can still occur in distilled water. However, any metabolic or developmental process involving free amino acids would be impaired because of their loss. The loss of amino acids seems to be dependent mainly on the absence of Na +, although osmotic pressure and ionic composition of the incubation solution seem to have some effect (Table 6). This observation is in accord with the requirement of .4rtemia for Na + for normal hatching and survival (Martin & Wilber, 1921; Boone & Baas-Becking, 1931 ; Croghan, 1958). The absence of Na + probably affects the cell membranes so that amino acids cannot be retained. For example, Rosenberg et al. (1965) have shown that Na + affects energy-independent (diffusion) as well and energy-dependent (active) transport of amino acids across mammalian intestinal tissue. Some of the amino acids appearing in the water may arise from emerged embryos which have burst. However, the large amounts and early appearance of amino acids in the water argue for the hypothesis that most of the amino acids in the water arise through disturbance of membrane transport phenomena. Incubation in 0.5 and 1.0 M NaCl. It is notable that glutamic acid, proline, and glycine have a similar pattern when Artemia are incubated in 0.5 M NaC1 (Table 2). All three amino acids increase steadily during the differentiation period and reach a peak at Ts0o/oE, after which they decline. The observation that these three amino acids are the same ones which are most affected by external osmotic pressure in some crustaceans suggests that the pattern observed in Artemia may be a response due to preparation for emergence. Most of the amino acid concentrations level off after T50% E in both 0.5 and 1.0 M NaCI (Fig. 1). The amino acid concentration is higher in 1.0 M NaC1, but the internal osmotic pressure caused by contributions from amino acids at the higher salinity is only slightly greater than in 0.5 M NaC1 (Fig. 2). This observation suggests that the increased level of free amino acids in response to higher external osmotic pressure has functions in addition to contribution to internal osmotic pressure. The nitrogen for the free amino acids in increased salinity may come from protein sources, since Dutrieu (I960) found decreased total protein N in animals incubated in greater salinity.

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Glutamic acid may have some special role in osmotic regulation, because it is present in smaller amounts in Artemia incubated in 1.0 M NaCI than in 0.5 M NaC1 (Tables 2-3). The role of glutamic acid may be related to the following discussion concerning glutamic dehydrogenase. The reasons for the free amino acid increase in response to external salinity are poorly understood. A mechanism involving glutamic dehydrogenase has been proposed to explain the higher amino acid levels in euryhaline species when adapted to higher osmotic pressures (Gilles &; Schoffeniels, 1964b; Schoffeniels, 1964). These authors studied glutamic dehydrogenase extracted from crayfish (Astacus fluviatilis) and lobster (Homarus vulgaris) muscle. They found that glutamic dehydrogenase activity depended upon the presence of monovalent cations (Na + or K+), and that the enzyme activity was higher in the euryhaline crayfish than in the stenohaline lobster. They proposed that the intracellular cation concentration affected glutamic dehydrogenase activity, which in turn regulated the concentration of the free amino acid pool. This concept is in accord with observations that changes in the cellular amino acid levels of the crab, Eriocheir sinemis, are mirrored by variations in the rate of nitrogen loss from the animal. Transfer from fresh water to sea water causes a temporary decrease in the rate at which nitrogen is lost during the period when free amino acids build up in the cells. Conversely, transference from sea water to fresh water is accompanied by a marked but transitory increase in the rate of nitrogen loss (Jeuniaux & Florkin, 1961). The time and rate of free amino acid buildup during Artemia development is modified by the external salinity. In 0.5 M NaCI, the period of free amino acid buildup starts immediately after hydration, and reaches a peak at 16 hr of development. In 1.0 M NaC1, the amino acid buildup starts at 16 hr and reaches a peak at 32 hr (Fig. 1). It is interesting to compare the rate of NH 3 excretion during these periods. In 0.5 M NaC1, the excretion rate is greater than for a comparable period in 1.0 M NaCI (Table 4). These observations suggest that the buildup of free amino acids in greater salinities may de partly due to decreased ammonia excretion. The rates of glutamic dehydrogenase activity are not significantly different at Ts0 % E for .4rtemia incubated in three salinities (Table 9). According to the hypothesis of Schoffeniels and his co-workers, it would be expected that enzyme activity would be higher for .4rtemia in greater salinities. Optimal conditions may not have been present for the assay of this enzyme, so that the measured enzyme activity may not reflect the true activity in .4rtemia. If Artemia does possess a glutamic dehydrogenase system responsive to the presence of monovalent cations, the requirement of Na + for normal hatching and survival would be explained in part. This topic is being studied in more detail. REFERENCES ALLENK. (1961) Amino acids in mollusca. Am. Zool. 1, 253-261. AWAPARAJ. (1962) Free amino acids in invertebrates: A comparative study of their distribution and metabolism. InAmlno Acid Pools (Edited by HOLDENJ. T.), pp. 158-186. Elsevier, New York.

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BELLINI L. (1957) Studio delle dipeptidase e proteinasi nello sviluppo di Artemia salina Leach. Rc. Accad. Lincei 22, 340-346. BELLINI L. (1960) Osservazioni sugli acidi nucleici nello sviluppo di Artemia salina Leach. Ricerca scient. 30, 816-833. BELLINI L. & LAVIZZAm G. S. (1958) Studio delle lipase nello sviluppo di Artemia salina, Leach. Rc. Accad. Lincei 24, 92-95. BELLINI L. & DE VINCENTIIS D. M. (1960a) Sulla eliminazione de ammonoiaca in Artemia salina Leach, diploide e tetraploide. Rc. Ist. Sci. Camerino 1, 60-66. BELLINI L. & DE VINCENTnS D. M. (1960b) Observations on the end products of protein metabolism in diploid and tetraploid Artemia salina (Leach). Expl Cell Res. 21, 239-241. BooNE E. & BAAS-BECKINGL. G. M. (1931) Salt effects on eggs and nanplii of Artemia salina L. aT. gen. Physiol. 14, 753-763. C ~ N P. S. (1956) Metabolic changes in free amino acids and peptides during urodele development. Expl Cell Res. 10, 675-686. Cn~N P. S. (1962) Free amino acids in insects. In Amino Acid Pools (Edited by HOLDEN J. T.). pp. 115-135. Elsevier, New York. CLEGG J. S. (1962) Free glycerol in dormant cysts of the brine shrimp Artemia salina, and its disappearance during development. Biol. Bull., Woods Hole 123, 295-301. C~GG J. S. (1964) T h e control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina, aT. exp. Biol. 41, 879892. CLEGG J. S. (1965) Biochemical and structural aspects of embryonic development in the crustacean, Artemia salina. Presented at the 132nd Meeting of the Am. Ass. Advanc. Sci., Berkeley, California, Dec. 26-31. COHEN P. P. & SALLACHH. J. (1961) Nitrogen metabolism of amino acids. In Metabolic Pathways. Vol. 2 (Edited by Gm~ENB~RQ D. M.). pp. 1-78. Academic Press, New York. CROCHAN P. C. (1958) T h e survival of Artemia salina (L.) in various media, ft. exp. Biol. 35, 213-218. DEUCHPa~ E. M. (1956) Amino acids in developing tissues of Xenopus laevis, jT. Embryol. exp. Morph. 4, 327-346. DEUCHAR E. M. (1962) T h e roles of amino acids in animal embryogenesis. Biol. Rev. 37, 378-421. DUTRmU J. (1960) Observations biochimiques et physiologiques sur le d~veloppement d'Artemia salina Leach. Archs. Zool. exp. gen. 99, 1-133. EMXRSON D. N. (1963) T h e metabolism of hatching embryos of the brine shrimp, Artemia salina. Proc. S. Dak. Acad. Sci. 42, 131-135. EM~TRSON D. N. (1966) Surface area respiration during the hatching of encysted embryos of the brine shrimp, Artenda salina. (In preparation). EMERSON D. N. & D ~ m ~ F. G. (1966) Some physiological effects of starvation in the intertidal prosobranch Littorina planaxis (Phillippi, 1847). Comp. Biochem. Physiol. T o be published. GARNER W. E. (1928) Osmotic pressure. In International Critical Tables of Numerical Data. Vol. IV, pp. 429-432. McGraw-Hill, New York. GILLES R. & SCHOF~NmLS E. (1964a) La synth~se des acides amin6s de la chalne nerveuse ventrale du homard. Bioehim. biophys. Acta 82, 518-524. GILLES R. & SCHOFFENIELSE. (1964b) Action de la v6ratrine, de la cocaine et de la stimulation 61ectrique sur la synth6se et sur le pool des acides amin6s de la chaL~e nerveuse ventrale du homard. Biochim. biophys. Acta 82, 525-537. HEILBRUNN L. V. (1956) An Outline of General Physiology. 3rd edn, p. 128. Saunders, Philadelphia. J ~ I A u x C. & FLORKIN M. (1961) Modification de l'excr6tion azot6e du crabe chinois au cours de l'adaptation osmotique. Arehs int. Physiol. 69, 385-386. KUTSKY P. B., EAKIN R. M., BERG W. E. & KAVANAUJ. L. (1953) Protein metabolism of the amphibian embryo. IV. Quantitative changes in free and non-protein amino acids..7. exp.Zool. 124, 263-278.

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LOCKWOODA. P. M. (1962) The osmoregulation of crustacea. Biol. Rev. 37, 257-305. MARTIN E. & WILBER B. (1921) Salt antagonism in Artemia. Am.J. Physiol. 55, 290-291. ]NTAKANISHIY. Y., IWASAKIT., OKIGAKIT. & KATO H. (1962) Cytological studies of Artemia salina. I. Embryonic development without cell multiplication after the blastula stage in encysted dry eggs. Annotnes zool. jap. 35, 223-228. OLSON J. A. & ANFINSENC. B. (1952) The crystallization and characterization of L-glutamic acid dehydrogenase. ~. biol. Chem. 197, 67-79. POTTS W. & PARRY G. (1964) Osmotic andIonic Regulation in Animals. pp. 158-163. Macmillan, New York. RIzzoLI C. (1957) Sulla composizione proteica ed aminoacidica dell' uovo di Trota durante lo sviluppo. Boll. Soc. ital. Biol. sper. 33, 223-226. ROSENBERGI. H., COLEMANA. L. & ROSENBERGL. E. (1965) The role of sodium ion in the transport of amino acids by the intestine. Biochim. biophys. Acta 102, 161-171. SCHOFFENIELSE. (1964) Cellular aspects of active transport. In Comparative Biochemistry. (Edited by FLORKIN M. & MASON H. S.). Vol. 7, pp. 137-202. Academic Press, New York. SELIGSON n . & SELIGSON H. (1951) A microdiffusion method for the determination of nitrogen liberated as ammonia. ~. Lab. din. Med. 38, 324-330. TROLL W. & CANNAN R. K. (1953) A modified photometric ninhydrin method for the analysis of amino and imino acids. 3t. biol. Chem. 200, 803-811. URBANI E. (1959) Protidi glucidi e lipidi nello sviluppo di Artemia salina Leach. Acta Embryol. Morph. Exp. 2, 171-194. WARNER A. H . & FINAMORE F. J. (1965) Isolation, purification, a n d characterization o f

p1, Pa-diguanosine 5'-triphosphate from brine shrimp eggs. Biochim. biophys. Acta 108, 525-530. WEISz P. B. (1947) T h e histological pattern of metameric development in Artemia salina, or. Morph. 81, 45-95. WHITAKER D. M. (1940) The tolerance of Artemia cysts for cold and high vacuum. J. exp. Zool. 83, 391-399.