Effect of anoxia on nucleotide metabolism in encysted embryos of the brine shrimp

DEVELOPMENTAL

BIOLOGY

27, 479-493

Effect of Anoxia

(1972)

on Nucleotide

Embryos of Biology,

in Encysted

of the Brine Shrimp’

D. M. STOCCO,~ P. C. BEERS, Department

Metabolism

University Accepted

AND

of Windsor, December

A. H. Windsor,

WARNER Ontario,

Canada

10, 1971

When encysted embryos of the brine shrimp, Artemia salinu, are maintained in an oxygen-free saline medium, morphogenesis and carbohydrate metabolism are arrested, but nucleotide metabolism continues in an orderly manner. Although anoxia inhibits the utilization of diguanosine 5’tetraphosphate (Gp,G) in encysted embryos compared to embryos exposed to air only, Gp,G is metabolized under anaerobic conditions and appears to be the primary source of phosphate-bond energy necessary to maintain the viability of anoxia-arrested embryos. We found that during 4 months of anoxia approximately 50% of the total available phosphate-bond energy in the embryo, mainly in the form of Gp,G, is utilized. Also, during prolonged periods of anoxia the adenosine nucleotide pools are depleted, but guanosine nucleotide metabolism continues. When anoxia is terminated by aeration all nucleotide pools, except for the diguanosine nucleotides, return to levels found in control embryos at the time of hatching. In addition, the rate of recovery of the nucleotide pools correlates well with the delay, if any, in the resumption of morphogenesis. Finally, as development of Artemia embryos approaches hatching, N, treatment appears to have a diminishing effect upon the control of guanosine nucleotide metabolism. INTRODUCTION

Extended periods of oxygen debt or anoxia during embryonic development usually produce morphological aberrations leading to cytolysis of the majority of developing animals. The deleterious effects of anoxia are often attributed to an accumulation of end products of anaerobic carbohydrate metabolism and/or to activation of acid hydrolases. The most common end-product is lactic acid, however some of the more primitive invertebrates accumulate pyruvic acid and/or glycerol when maintained in the absence of oxygen (Saz and Lescure, 1966; Von Brand, 1946). The brine shrimp, Artemia dina, offers an interesting exception to these observations and therefore has been the subject of several studies on the effects of anoxia on embryonic development. In a previous study Ewing (1968) demonstrated that morphogenesis of ‘Research supported by the Council of Canada (Grant A-2909). 2Present address: Department versity of Toronto, Canada.

National

Research

of Zoology,

Uni-

encysted gastrulae of Artemia is arrested when the embryos are placed in an O,free medium, and in a separate study, Dutrieu and Christia-Blanchine (1966) reported that hydrated Artemia embryos are able to withstand at least 5 months of anoxia with little effect upon the carbohydrate and lipid reserves or upon subsequent development. More recently, Ewing and Clegg (1969) confirmed these findings and further indicated that they were unable to measure any appreciable changes in the common end products of anaerobic carbohydrate metabolism of encysted gastrulae of Artemia maintained in a pure N, atmosphere for 8 hr. These findings suggest that either energy metabolism is not required to maintain the integrity of developmentally arrested Artemia embryos, or an alternative energygenerating mechanism exists to support whatever metabolic activity is necessary to maintain the embryos in a viable state during prolonged periods of oxygen deficiency. In two separate studies Clegg et al.

479 Copyright

0 1972 by Academic

Press, Inc.

480

DEVELOPMENTAL

BIOLOGY

(1967) and Warner and McClean (1968) demonstrated that the nucleotide anhydride P’, P4-diguanosine 5’-tetraphosphate (Gp,G or diGDP) is an important source of cellular purines in Artemia, and suggested that Gp,G may also function as a source of ATP and/or phosphate-bond energy for development. In view of these findings we undertook a study on the effect of anoxia on nucleotide metabolism in Artemia embryos in an attempt to understand how these embryos are able to withstand prolonged periods of oxygen deficiency. The results of this study are presented here and indicate that nucleotide metabolism, unlike carbohydrate metabolism, is not static during periods of anoxia. Marked changes occur in the levels of the adenosine nucleotides and in the diguanosine nucleotides (Gp,G and Gp,G), whereas only slight changes occur in the guanosine mononucleotides during nitrogen treatment. Although the changes in adenosine nucleotides in Artemia are similar to those reported for other embryonic systems maintained anaerobically (Barth and Jaeger, 1947; Brachet and Ledoux, 1955), cytolysis does not occur in hydrated embryos of Artemia even after several months of anoxia, and the effects of anoxia are reversible. MATERIALS

AND

METHODS

The experimental results reported here were obtained during a three year period using two separate batches of dried encysted embryos of the brine shrimp, Artemia salinu, from the salterns in Utah (Sanders Brine Shrimp Co., Ogden, Utah). Batch 1 was obtained from Utah in August 1967 and batch 2 was obtained in December 1969. Although both batches were obtained from Utah, we found them to be different in several respects. Batch 1 gave about 70% hatch shortly after their arrival, whereas batch 2 gave about 45% hatch. Their responses to anoxia were similar in most ways except that the

VOLUME

27, 1972

changes in the nucleotide levels, particularly in the adenosine nucleotides, occurred much more rapidly in embryos from batch 1 than in embryos from batch 2. Incubation procedures. The encysted embryos were sterilized by immersion in 7% antiformin solution for 15-30 min at 4°C as previously described (Nakanishi et al., 1962). Following removal of floating cysts and debris by suction, the remaining cysts were collected and washed several times with cold distilled water on a fritted-glass filter. One-gram portions of sterile cysts (about 100,000 embryos) were weighed directly into 250-ml Erlenmeyer flasks and covered with 50 ml of ice-cold sterile sea water supplemented with penicillin (1000 units/ml) and streptomycin sulfate (100 pg-ml) as previously described (Warner and McClean, 1968). The flasks containing the cysts and sterile medium were kept in an ice-bath until needed. Development of embryos from both batches was initiated by immersion of the flask in a 30°C bath, and the flasks were agitated gently throughout the experimental period to ensure an adequate oxygen supply. The samples used in the anaerobic studies were purged continuously with purified nitrogen for at least 1 hr, and the flasks were stoppered tightly. For the anaerobic study encysted short-term embryos from batch 1 were used exclusively, and the flasks containing these embryos under N, were maintained at 30°C with gentle shaking until needed. For the long-term anaerobic study, embryos from batch 2 were used exclusively and the stoppered flasks were maintained without shaking at 22-24°C. (In one experiment the flasks containing an N, atmosphere were maintained in a large desiccator containing pure N, and alkaline pyrogallol but no differences were noted compared to stoppered flasks maintained separately; therefore, this procedure was discontinued.) The nitrogen (prepurified grade, Matheson) was further purified by

STOCCO, BEERS, AND WARNER

Nucleotide

bubbling through a series of four flasks containing: (1) 7.5 N NH,OH saturated with NH,Cl and containing copper filings, (2) N H&SO, containing 2 drops of a 1% phenolphthalein solution, (3) distilled water, and (4) sterile sea water. Isolation of nonviable encysted embryos of Artemia. Every batch of commercially

available brine shrimp eggs contains large amounts of nonviable encysted embryos (20-60s) which cannot be separated from the viable ones using sedimentation techniques employing organic or aqueous media (Warner, unpublished observations). Since the changes in nucleotide levels under anaerobiosis could be the result of degradative changes occurring in the nonviable embryos only, we devised a method of collecting these embryos and testing this possibility. About 20 g of cysts from batch 2 were sterilized with antiformin and incubated in air at 30°C in sterile sea water fortified with penicillin and streptomycin sulfate as described before. At 24-hr intervals over a 4-day period the embryos that emerged or hatched along with floating empty shells were removed and the remaining encysted embryos were resuspended in fresh sterile sea water. At the end of 4 days the encysted embryos that remained were collected on a fritted-glass disk, washed well with distilled, water, and used immediately for the anoxia study. Extraction and purification of acidsoluble purine-containing compounds. At

varying times the contents of the flasks were collected on a fritted-glass filter and washed thoroughly with distilled water. The cysts were transferred quantitatively to a tissue grinder (Ten Broeck type) and homogenized in 25 ml of ice-cold N HClO,. The homogenates were centrifuged at 12,000 g for 15 min and the acid-soluble fraction was retained. The acid-insoluble pellet was washed’once with 10 ml of icecold 0.5 N HClO, and the soluble fraction, collected by centrifugation, was combined with the first soluble fraction. The

Metabolism

481

in Artemia Embryos

combined acid-soluble fractions were deacidified by shaking with Alamine and the nucleotides were fractionated on 1 x 40 cm columns of DEAE-cellulose (DE-23, described Whatman) as previously (Warner and Finamore, 1967). When necessary the AMP fraction eluted from DEAE-cellulose was further purified on columns of Dowex-l-Cl-, 2%, using a chloride system (Cohn, 1950). This step was necessary to separate AMP from UMP, ascorbic acid sulfate, and a derivative of UDP which elute with AMP. The free purines and purine-containing nucleosides which come directly through the DEAEcellulose column were purified as follows. The DEAE-cellulose fraction containing the purine bases and nucleosides was evaporated to dryness under reduced pressure and the residue was dissolved in 5 ml of 2 N HCl. To determine the total adenine and guanine content, the samples were heated for 1 hr at lOO”C, cooled, then applied to columns of Dowex-50-H+, 8%, and developed with 2 N HCl according to Cohn (1955). When the content of guanosine and adenosine was desired, the sample in 2 N HCl was applied directly to the cation-exchange column without prior heating and developed with 2 N HCl as before. The W-absorbing fractions were identified using 2801260 ratios and on the basis of their elution sequences as described previously (Warner and Finamore, 1967; Warner and McClean, 1968) and quantitated using the proper extinction coefficients (Volkin and Cohn, 1954; Finamore and Warner, 1966). Whenever possible, standard deviations about the mean were determined. The number of cysts, prenauplii, and/or nauplii in each flask was determined as previously described (McClean and Warner, 1971). RESULTS

The Effect of Anoxia on Resumption of Development in Artemia Embryos

Dutrieu

and Christia-Blanchine

(1966)

482

DEVELOPMENTAL

BIOLOGY

reported that hydrated encysted embryos of Artemia salina are able to withstand at least 5 months exposure to an N, atmosphere then resume development normally upon aeration. In this study we observed that although the initial rates of emergence and hatching of Artemia embryos from batch 2 maintained in pure nitrogen for periods up to 4 months are similar to rates for control embryos, the minimum time for the onset of emergence and hatching increases as a result of anoxia. These results are shown in Table 1. These data also clearly show that the delay in onset of emergence and hatching is not directly porportional to the time spent under N, but reaches a plateau around 40 days. In contrast to these findings we observed that embryos from batch 1 recovered very rapidly and without delay after short periods of anoxia (24 hr). In this respect these embryos appear to be similar to those used by Dutrieu and ChristiaBlanchine (1966). Aerobic versus Anaerobic Nucleotide Metabolism

Guanosine

In an earlier report, Warner and Finamore (1967) indicated that development of prenauplii (in air) is associated with a decrease in the amount of embryonic GMP, GDP, and Gp,G, whereas the quantity of GTP increases. When hydrated encysted embryos of Artemia from batch 1 were incubated in a N, atmosphere and compared to embryos incubated in air, strikingly different results were obtained. From the data in Table 2 it is apparent that the utilization rates of GMP and Gp,G are suppressed during incubation in N,, whereas GDP utilization and GTP production are not affected initially (O-5.5 hr) but only after additional incubation under N, (5.5-12 hr). Moreover, the amount of GMP and diguanosine 5’-triphosphate (Gp,G) increases under anaerobic conditions compared to controls (0 hr) and/or embryos incubated in air for an identical period of time.

VOLUME

27, 1972

TABLE 1 INFLUENCE OF ANOXIA ON EMERGENCE HATCHING OF Artenia EMBRYOS’

0 3 8 18 42 116 218

AND

9.0

1.0

15.0

1.0

100

13.0 15.0 16.7 17.3 ND” ND

1.0

18.0 19.5 22.3 23.0 ND ND

0.9 1.0 1.0 1.0 ND ND

>99 98 ND >94 102 74

0.9 1.0

0.9 ND ND

“These embryos were taken from batch 2. The time required for the onset of emergence and hatching was determined at 30°C as described in Materials and Methods. b Bate of emergence of embryos maintained under N, for the times indicated (E), compared to the rate of emergence of embryos exposed to air only (C) following the onset of emergence. u Bate of hatching of embryos maintained under N, for the times indicated (E), compared to the rate of hatching of embryos exposed to air only (C) following the onset of hatching. d Not measured.

When embryos from batch 2 were maintained under N, for extended periods (up to 4 months) and the guanosine nucleotides were analyzed as before, results similar to those for embryos from batch 1 were obtained. These data are summarized in Table 3. It is important to note that although morphogenic activity is suspended under anoxia, the concentration of Gp,G in these embryos declines throughout the 4 months in N,, whereas the level of Gp,G remains constant after increasing to a maximum around 30 days. In addition, the initial rapid decrease in the levels of GDP and GTP in response to anoxia is followed by a more gradual decrease in the levels of these nucleotides. Also, the level of GMP declines under anaerobic conditions but not until it has reached a maximum at about 14 days. Finally, it was observed that the level of Gua plus Guo increases steadily throughout the period of anoxia, and the increase in these compounds is concomitant with the overall decrease in the guanosine-containing nucleotides.

STOCCO,

BEERS,

AND

Nucleotide

WARNER

Metabolism

TABLE EARLY

CHANGES

IN NUCLEOTIDE

LEVELS

483

2

Artemia

IN

in Artemia Embryos

EMBRYOS

UNDER

AEROBIC

AND

ANAEROBIC

CONDITIONS” rMoles

per 1OO.O(Ml embryos

i SD”

COmpWllld

0 Hr

2 Hr N,

2 Hr D

,iSHrD

3.s Hr N,

12HrD

GMP

‘2.88 * 0. :15

2. 18 i 0.w

.A.14 * 0.11

2.0”

f 0.29

9.07

l

0

ADP

0.37 t 0.01

0.2:i i 0.01

0 29 z 0.04

0.a

* 0.M

GDP ATP GTP G&G Gp,G

2.4% i 0.16 0.07 * 0.09 0.98 * 0.16

1.99 IL69 1 .:ii 1.19 6.84

1.90 0 :x5 1 :is 1.43 i.13

1.9” 0.7’ I.40 1 11 6.63

1 L * * 2

0.:14 2.12 O.28 1.26 1.28 6 i.5

+ t T i

0.08 0.15 0.07 0.18 0 07 0 28

1 .I4

l

0.03

i :a1 IO.29

i * I i i

0 0.01 0.0; 0.09 0.24

“These embryos were taken Srom batch 1. At the times Materials and Methodb. b Standard deviation of the mean: n Zi. ’ Development in air.

x z * =t i

0.14 0.07 0.03 0.17 0.34

indxated

the acid-soluble

TABLE CHANGES

Compound

IN NLJCLEOTIDE

LEVELS

o.txj 0 10 0 22 0 04 0.13

IN HYDRATED

were extracted

* i + i i i i

4 07 f 0.55

0.2 0. 13 0.26 0.07 0.12 0.04 0.21

0 “8 2 09 n 1.5 1.11 1 “7 6.48

and purified

2 0.09 IO.01 L 0.14 l 0.06 2 0.13 IO.48

ah described

GASTRULAE

per 100,000

OF

Artemia

MAINTAINED

IN AN

embryos 110 Days N2

0 Day

3 Days

7 Days

14 Days

31 Days

56 Days

Gua + Guo AMP GMP ADP GDP ATP GTP

3.03 0.41

3.35

3.65 0.51

4.05 0.58

5.02 0.48

6.07

1.52

2.03 0.25

2.12

2.48 0.05

2.09

0.18

0.05

0.10 1.85
0.97

0.87

0.84

0.82

0.75

0.77

0.44 1.40

0.38

0.22 0.71





0.56

0.56

0.40

0.30

GP~G

0.80

1.53

2.28

2.48

GP,G

7.35

6.25

5.10

4.50

2.44 3.73

3.09

0.34 1.66

“These embryos were Material and Methods.

0.95

taken

from

1.93 5.90 batch

versus Anaerobic Adenosine Nucleotide Metabolism

Aerobic

2 and

the

in

3

ENCYSTED N 2 ATMOSPHERES

PMoles

nucleotides

1.79 0.29 1.59 O.i4 1.98 1 14 5.67

03

12 Hr N,

acid-soluble

n’ucleotides

prepared

7.18 <0.05 1.41


2.44

as described

in

These data are shown in Table 2. (Owing to technical difficulties in this experiment the AMP content was not measured Previously Warner and Finamore (1967) accurately and therefore is not reported.) demonstrated that ATP production When hydrated embryos from batch 2 occurs rapidly in encysted embryos of were maintained under N2, the levels of Artemia following resumption of developADP and ATP were observed to decline ment in air. From their study they also slowly over a 14-day period, whereas the suggested that synthesis of ATP occurs level of AMP increased slightly to day 14 partly at the expense of preexisting AMP then declined to an undetectable level and ADP, and partly at the expense of by day 110. Also, we observed that when another purine nucleotide(s). When these embryos were maintained under N, encysted embryos from batch 1 were for extended periods of time, the level of incubated in N,, the initial response the adenosine nucleotides declined with(O-5.5 hr) was an increase in the ATP out a concomitant increase in the level level to 50% of the level in control of adenosine and/or adenine. embryos accompanied by a similar deIn another series of experiments using crease in ADP. In contrast, prolonged Artemia cysts from batch 1, the embryos incubation in N, (5.5-12 hr) permits a were permitted to develop in air for 5.5 decrease to occur in both ADP and ATP. hr at 30°C then purged continuously

484

DEVELOPMENTAL

BIOLOGY

with N,. (In embryos from batch 1 the onset of emergence of prenauplii occurs at 5.5 hr.) Embryos maintained in this way were collected at 0.5, 1, and 5 hr after initiation of the N, treatment and the adenosine nucleotide levels were determined. Another group of embryos from the same batch was reaerated after 5.5 hr incubation in air and 24 hr in N, and the same nucleotides were assayed at 5, 15, and 30 minutes after termination of anoxia. The results of these experiments are shown in Fig. 1. It is important to note that ATP is rapidly and completely depleted in these embryos following transfer to a pure N, environment, whereas ADP declines more slowly. Also, the decrease in ADP and ATP is accompanied by a concomitant increase in AMP in these embryos. When the embryos were returned to an aerobic environment the original levels of ADP and ATP were restored rapidly and completed within 5 and 15 min, respectively. It is

VOLUME

27. 1972

noteworthy that ATP “overshoots” the control level following aeration, and the rise in ADP and ATP occurs entirely at the expense of preexisting AMP. In a similar experiment carried out with Artemia embryos from batch 2 no changes in ADP and ATP were detected after several hours in N,. Subsequently it was observed that anoxia lowers the levels of ADP and ATP in embryos from batch 2, but the rate of decline is extremely slow compared to the rate for embryos from batch 1. The rate of recovery of ADP and ATP following aeration was equally slow, requiring several hours to return to levels found in control embryos. Nucleotide Embryos

Metabolism in Artemia after Extended Periods of

Anoxia Since embryos from batch 2 were found to respond slowly to conditions of anoxia compared to embryos from batch 1, we decided to examine the rates of recovery

I

‘1.

ATP

/

5

HOURS

IN AIR

HOURS

IN NITROGEN

FIG 1. Aerobic versus anaerobic adenosine nucleotide metabolism 1 were permitted to develop in air up to 5.5 hr at 3O”C, purged with 30 min at 30°C. The acid-soluble adenosine nucleotides were isolated Materials and Methods.

30

I5

MINUTES

IN AIR

in Artemia embryos. Embryos from batch N, for up to 24 hr, then aerated for up to as indicated and purified as described in

STOCCO, BEERS, AND WARNER

Nucleotide

of all acid-soluble nucleotides in these embryos at a time when the reserves of ADP and ATP were exhausted. Fully hydrated encysted gastrulae were maintained in a pure N, atmosphere at room temperature. After 26 days of anoxia, the embryos were aerated and maintained with gentle shaking at 3O”C, and samples were taken for nucleotide analysis at 0, 1.5, 5.5, 21, and 30 hr. The results of this experiment are summarized in Table 4 and compared to embryos exposed to air only (controls). It is apparent that the recovery of ADP and ATP following termination of anoxia proceeds much slower in these embryos compared to embryos from batch 1, but otherwise, a similar pattern is evident. Also, it should be noted that the concentrations of all purine-containing compounds, except for the diguanosine nucleotides, return to control levels prior to the onset of hatching. There is a tendency for Gp,G and Gp,G levels to return to control levels following cessation of anoxia, but this does not occur to any appreciable extent. These results along with those in Table 3 are summarized graphically in Fig. 2. (It is important to note that the onset of hatching in embryos RECOVERY

OF NUCLEOTIDE

LEVELS IN Artemia

Metabolism

The Effect of N, on Nucleotide Levels in Artemia Embryos Administered before/after Resumption of Development

The observation that Artemia embryos are able to resume development after prolonged exposure to anaerobic conditions prompted us to examine the acid-soluble nucleotides in hydrated embryos either pretreated with N, or whose development was interrupted by N,. In one experiment, hydrated cysts from batch 1 were maintained under N, for 24 hr at 22-24”C, then aerated and permitted to develop to 5.5 or 12 hr at 30°C at which time the acidsoluble fraction was prepared and analyzed. In a second experiment, Artemia cysts from batch 1 were allowed to develop for 5.5 or 12 hr in air then purged with N, and maintained anaerobically for an additional 24 hr before subjecting the nucleotide fraction to analysis. The results of both experiments are compared in Table 5. In general, preincubation in N, retards slightly the utilization of GMP and GDP

TABLE 4 EMBRYOS IN AIRY

FOLLOWING per 100,000

ANOXIA

15 Hr

TO EMBRYOS

Experimental’ 24 Hr

0 Hr

1.5 Hr

5.5 Hr

21 Hr

30 Hr

5.3 0.18 1.58 0.26 0.95 0.30 1.01 2.37 4.20

4.3 0.22 1.31 0.49 0.96 0.63 1.32 2.34 4.53

3.5 0.26 1.27 0.14 1.02 1.07 1.65 2.28 5.18

3.9 0.29 1.22 0.21 1.15 0.86 1.56 2.25 5.00

3.0 3.0 2.9 4.6 0.41 0.27 0.33 0.57 1.52 1.28 1.25 2.16 0.34 0.29 0.30 0.04 1.66 1.29 1.09 0.85 0.44 0.68 0.81 <0.05 1.40 1.32 1.46 0.60 0.80 1.07 1.30 2.40 7.35 7.05 6.62 4.60 were taken from batch 2 and the acid-soluble and Methods. were exposed to air only, and under these

conditions

were

then

maintained

COMPARED

embryos

Control” 0 Hr

Gua + Guo AMP GMP ADP GDP ATP GTP GP~G GP,G a These embryos scribed in Materials *These embryos 12 hr at 30°C. c These embryos indicated.

485

Embryos

maintained anaerobically for 26 days is delayed several hours and occurs at 22.5 hr at 30°C.)

FMoles Compound

in Artemia

anaerobically

for

26 days,

fraction

was purified

aerated

the onset and

and analyzed of hatching

maintained

as deoccurs

at 30°C

at as

HOURS

IN AIR

x AMP

----)t -0-

” 4

7

0 ADP . ATP Gu’n G:O

5,

A:,

_

,* A-----~ :

XjX

-

*-

/“\

/

\ “\ \ x>-Mf \ \ x--4--

\

l\:d.

‘Pb,

/ xv-

/

r-

/ -,--o-

,o/oL-\

-,-.

--__

70x

-0-

.? II

-----.

GD,P GT!

I

6

4

?

-g--

/o /o to /

1 0

I

-L--oAL--o

G$

-I,

IO

20

30

DAYS

50

40

UNDER

FIG.

60

’

60

100

120

NITROGEN

2. Changes in the acid-soluble purine-containing compounds in Artemia embryos in response to anoxia (--) and subsequent development in air (-----). Embryos from batch 2 were used exclusively and the acidsoluble nucleotides were prepared and purified as described in Materials and Methods. After 26 days of anoxia some of the embryos (100,000 per sample) were aerated then permitted to develop in air up to 30 hr at 30°C. The symbols --x-, -O-, -0--, and -Arepresent nucleotide levels of control embryos just after hatching (15 hr) exposed to air only. 486

Nucleotide Metabolism

STOCCO, BEERS, AND WARNER

487

in Artemia Embryos

TABLE 5 EFFECT OF

N,

ON NLJCLEOTIDE

LEVELS IN

Artemia EMBRYOS BEFORE/AFTER RESUMPTION 24HrN,

AMP GMP ADP GDP ATP GTP Gp,G GP.G

0 Hr

,S.S Hr D

2 8X 1 0.3s

0.16 2.w 1 O.“Y 0.n f 0.0~

0.37 2.42 0.07 0 98 1 14 7.31

* I + * i i

S.5 Hr D

S.5 hr D

0.01 O.Ifi 0.09 O.lfi 0.03 0.29

1.9-e 0.72 1 .40 1.11 6.64

T f i L t

0.06 0.30 0.22 0.04 0.13

I.93 I).@ 2. ni 0.67 I.45 1.40 5.64

‘4h:N,

ND” * o.s:i i 0 17 t 0, :19 10 ‘9 1 0.1s * 0.19 + 0.X

‘) These embryos were taken from hatch 1 and the acid-soluble ‘Standard deviation of the mean: n 3. ’ Development in air nnlv ‘I Not measured.

12 HI D

ttaction

(compared to controls), and enhances the level of ATP at both stages examined. Pretreatment of encysted gastrulae had little effect upon ATP production during subsequent aerobic incubation except for the 12-hr stage. Also, it is clear that pretreatment with N, enhances the level of Gp,G and stimulates the utilization of Gp,G at all stages examined. When development of late gastrulae and prenauplii (5.5- and 12-hr embryos, respectively) is interrupted by N, a somewhat different pattern emerges. It appears that as development approaches the hatching stage N, treatment has a diminishing effect upon the utilization of GMP and GDP, whereas GTP “synthesis” is markedly inhibited. Examination of the levels revealed adenosine nucleotide that ADP and ATP become undetectable within 24 hr following interruption with N,, whereas the AMP content rises. The loss in ADP and ATP from these embryos (batch 1) can be accounted for entirely by the increase in AMP (see also Fig. 1). Since it was observed that Artemia embryos from batch 2 responded somewhat differently to anoxia, embryos from this batch were interrupted at two stages of development by N, treatment and their nucleotide profiles compared to profiles from batch 1 embryos. In one experiment embryos were allowed to develop for 9 hr at 30°C purged with N,, then maintained

I. 10 I.67 2.01 1,li I.33 fi.oe

* II.45 0 * o.so 0 10.17 * 0.19 * 0.2s

was prepared

I .79 0.29 1 ..i9 0.74 1.38 1.14

ND i Cl.22 IO.13 r 0.30 10.07 e 0.12 7 0.04

5.67

and analyzed

l

0.21

as described

OF DEVELOPMENT”

“4 Hr K, t 12 hr D ND 2.13 + 0.65 I LR9 l 0.34 i 0.97 h

12 Hr D “4h+rN., ND 7 0.x

0.05 0.24

“.oi

n.4.5

1 :,9 7 0 1.06 i 1.15 f .3.3R t

0. 13 0.1.5

1.22 + 0.21 R.:IS + 0.42 in Materials

0.32 0.n 0.10 0.69

and Meth&

under N, for varying durations up to 8 days. At the point of interruption (9 hr) these embryos had reached the same developmental stage as the 5.5hr embryos from batch 1 and therefore they should be comparable. In another experiment embryos from batch 2 were allowed to develop for 24 hrs and the nauplii and prenauplii that resulted were treated with N, and maintained under pure N, for up to 5 hr. These embryos were found to be more susceptible to the deleterious effects of anoxia then encysted embryos; therefore, they were maintained under N, only as long as they were able to completely recover upon subsequent aeration (see Ewing and Clegg, 1969). The changes in nucleotide levels in these embryos in response to anoxia are shown in Table 6. In general the data obtained for embryos from batch 2 were similar to those obtained using embryos from batch 1. However, it should be noted that the changes in the adenosine nucleotide pools occur much slower in embryos from batch 2 than in embryos from batch 1. In addition, it appears that the rate of utilization of GTP and Gp,G in prenauplii and nauplii is markedly inhibited by anaerobiosis, whereas this is not the case with younger embryos. Finally, it was observed that the loss of ADP and ATP from these embryos cannot be accounted for entirely by a rise in AMP, adenosine, and/or adenine.

488

DEVELOPMENTAL

EFFECT

Compound Gua + Guo AMP GMP ADP GDP ATP GTP

GP,G GP,G

“These terials and b These c These

OF ANOXIA

ON NUCLEOTIDE

PMoles

BIOLOGY

LEVELS

per 100,000

VOLUME

TABLE

6

IN LATE

GASTRULAE

9-hr

1 Day

2.90

3.14

0.36

0.31

4.5 Days 3.57 0.36

1.23

1.37

1.45

1.58

0.44

0.29

0.26

0.26

0 Hr

3.91

4.30 0.40 0.73 0.60 1.09 1.27 1.56 0.58 4.93

0.30

1.25

1.03

1.02

0.98

0.69

0.30 1.03 1.65 6.00

0.15 0.91 1.94 5.52

1.46

1.32 6.17

NAUPLII

8 Days

0.76 1.50

AND

PMoles

embryo@

0 Day

0.95 6.75

27, 1972

OF Artemia

per 100,000

embryos were taken from batch 2 and the acid-soluble nucleotides prepared Methods. embryos emerge as prenauplii beginning at 9 hr in air at 30°C. samples each contained approximately 60% nauplii and 40% prenauplii.

The fate of the adenosine nucleotides under conditions of anoxia remains to be ascertained. Effect of Anoxia on Nucleotide Levels in Nonviable Embryos The observation that all commercially available Artemin cysts contain nonviable embryos which are difficult to separate from viable encysted gastrulae, and the possibility that the changes observed under N, might be occurring in the nonviable embryos only, prompted us to investigate whether or not the nonviable embryos in the population were responsible for the changes measured under anoxia. The data in Table 7 indicate that only slight changes occur in the nucleotide levels of nonviable embryos under N,; therefore, the nucleotide profile observed in Artemia embryos in response to anoxia must be due primarily to changes in the viable cysts. It is evident that differences in the nucleotide levels exist between nonviable embryos and the total population of embryos (prior to incubation in NJ ; however, it is not yet certain whether these differences are characteristic of unincubated nonviable embryos, or arise during the 4-day period needed to harvest nonviable embryos.

salina” 24-hr

embryos’

2 Hr 4.74 0.31 0.84 0.26 0.79 1.03 1.72 0.67 5.30 as described

5 Hr 4.76 0.32 0.94 0.38 0.91

0.85 1.55 0.71 5.27 in Ma-

DISCUSSION

The ability of the brine shrimp, Artemia salinu, to survive in brackish ponds and highly concentrated brines where the oxygen content is often immeasurable is well known. Moreover, it has been clearly demonstrated that fully hydrated encysted embryos of Artemiu are able to withstand prolonged periods of anoxia at room temperatures without alteration in their gross morphological pattern then to resume development normally upon aeration (Dutrieu and Christia-Blanchine, 1966; Ewing, 1968; Ewing and Clegg, 1969). The mechanism(s) which permit(s) Artemiu embryos to maintain their cellular and developmental integrity under conditions of anoxia is not yet understood, but Dutrieu and Christia-Blanchine (1966) and Ewing and Clegg (1969) both reported that carbohydrate metabolism is quickly brought to a standstill by anoxia. The latter investigators also reported that the common end products of anaerobic carbohydrate metabolism do not accumulate under conditions of anoxia. If carbohydrate metabolism in Artemia embryos is kept in check during periods of anoxia, the mechanism(s) by which these embryos provide the energy to maintain a viable but morphologically

STOCCO,

BEERS,

TABLE EFFECT

OF ANOXIA

PMoles

Gua + Guo AMP GMP ADP GDP ATP GTP GP& GP,G

Control”

3.0 0.41

1.52 0.34 1.66 0.44 1.40 0.80 7.35

Nucleotide

WARNER

7

ON THE NUCLEOTIDE

IN NONVIABLE

Compound

AND

LEVELS

EMBRYOS”

per 100,000

embryos

Nonviable encysted embryos ‘O$ 2

Nonviable encysted embryos (7 days NJ

4.0 0.45 1.32 0.16 0.97 0.35 1.08 1.76 5.50

4.4 0.45 1.15 0.13 0.87 0.35 0.81 1.85 5.25

a See Materials and Methods regarding collection of these embryos from batch 2. h These values were obtained from the total population of encysted embryos; i.e., viable plus nonviable embryos from batch 2.

inactive state poses an interesting problem. In 1963 Finamore and Warner reported that Artemia embryos contain large amounts of a unique nucleotide anhydride, P I, P 4-diguanosine 5’-tetraphosphate (Gp,G), and at that time they suggested that this compound may serve as a primary source of phosphate-bond energy during development. In subsequent studies, Clegg et al. (1967) and Warner and McClean (1968) demonstrated that Artemiu are unable to synthesize purines de nova, and indicated that Gp,G is the major, and perhaps only, source of purinecontaining nucleotides in the developing embryo. A more recent report has indicated that Gp,G may participate in the control or regulation of DNA synthesis during development in Artemia (Finamore and Clegg, 1969). In view of the importance of Gp,G during development, we deemed it mandatory to study nucleotide metabolism under conditions of anoxia in an attempt to ascertain whether or not anaerobic nucleotide metabolism is able to provide the energy necessary to sus-

Metabolism

in Artemia

Embryos

489

tain the viability of morphogenetically arrested embryos. A thorough understanding of nucleotide metabolism in Artemiu in response to anoxia has proved difficult due to the following observations. First, encysted embryos of Artemia sulinu are impermeable to all radiolabeled nucleotide precursors except ‘%-bicarbonate, and the fact that these embryos do not synthesize purines de novo has prevented us from using radiolabeled nucleotide precursors in this study (Clegg et al., 1967; Warner and McClean, 1968). Second, we have observed that commercially available Artemia cysts obtained over a two-year period from the supplier in Utah respond differently to both aerobic and anaerobic conditions. We found that under optimal conditions embryos from batch 2 required a longer time to emerge (9.0 versus 5.5 hr), took longer to hatch (15 versus 12 hr), and generally responded slower metabolically to aerobic conditions compared to embryos from batch 1. On the other hand, cysts from batch 1 responded to anoxia in a manner similar to that described by Dutrieu and Christia-Blanchine (1966), whereas cysts from batch 2 responded more slowly to conditions of anoxia as measured by the rate of change in the nucleotide profile and recovery time following termination of anoxia. It should be pointed out, however, that the overall changes in nucleotide metabolism, measured either aerobically or anaerobically, were similar with embryos from both batches and that only the time required for these changes to occur differs. When hydrated encysted embryos of Artemia are maintained in an oxygen-free atmosphere nucleotide metabolism, unlike carbohydrate metabolism, is not suspended (see Table 3 and Fig. 2). In fact, we noted that the level of Gp,G decreases steadily in Artemiu cysts maintained anaerobically, whereas the level of Gua plus Guo increases. Also, the concen-

490

DEVELOPMENTAL

BIOLOGY

VOLUME

trations of GDP and GTP decline by about 50% during the first week of anoxia then remain relatively constant, and the concentrations of ADP and ATP decrease to immeasurable levels during the first 2 wks. From these data we determined that during the first 4 months of anoxia, approximately 50% of the total available phosphate-bond energy, mainly in the form of Gp,G, was utilized and of this amount 50% was utilized during the first 2 wks. These data summarized in Table 8. We conclude, therefore, that diguanosine tetraphosphate is the main source of phosphate bond energy during periods of anoxia and that the energy requirements needed to suspend morphogenesis are much greater than the requirements needed to maintain the state of morphological inactivity once this state has been reached. The role of the enzyme, P’,P4-diguanoasymmetricalsine 5’-tetraphosphate pyrophosphohydrolase (asym-diGDPase), must also be examined if Gp,G is of primary importance as an energy source. In a previous study, Warner and Finamore (1965) demonstrated that asym-diGDPase is the only enzyme in Artemia cysts able to hydrolyze Gp,G, and they identified the products of hydrolysis to be GMP and GTP in equimolar amounts. It follows, then, that during the period of rapid Gp,G utilization an increase in GMP and GTP should result. That this is not entirely

CHANGES Source of phos-

phate group

-PO, per mole of nucleotide

ADP GDP ATP GTP GPZ GP,G 0 These

IN PHOSPHATE-BOND

values

CONTENT

21. 1972

the case is obvious from Fig. 2. Although GMP increases during the first 2 wks of anoxia, the level of GTP decreases. Perhaps the GTP that is liberated from Gp,G is metabolized rapidly to GMP then to Guo, and the energy liberated from the pyrophosphate linkages is utilized to maintain the embryos in a viable state. The role of diguanosine triphosphate (Gp,G) during development is not known, but we have observed that the level of this constant compound either remains (Warner and Finamore, 1967) or increases slightly prior to hatching (see Table 4) depending upon the source and batch of embryos. Under conditions of anoxia the level of this compound increases 3-fold during the first month then remains constant thereafter. The reason for this increase and the role of Gp,G during anoxia remains to be elucidated. The mechanism by which Artemia embryos inactivate glycolysis in response to anoxia is still unknown, but may depend upon the proper ratio of AMP, ADP, and ATP to one another and/or to the guanosine nucleotides (Ishihara and Kikuchi, 1968). When encysted embryos are maintained for prolonged periods of anoxia (several months) the levels of all adenosine nucleotides become immeasurable (using our techniques). If development in Artemia is interrupted just prior to emergence by removal of oxygen from the incubation medium, the levels of ADP and ATP de-

TABLE 8 IN Artemia PMoles

-PO,

EMBRYOS

IN RESPONSE TO ANOXIA*

per 100,000 embryos

0

3

7

14

31

56

110 days N*

1

2.00

1.22

1.05

0.87

0.86

0.87

0.78

2

5.36

5.72

5.72

5.70

6.08

5.70

5.66

3

~~~~~~22.05 29.41

18.75 25.69

17.70 24.47

15.33 21.90

13.50 20.44

11.20 17.77

9.31 15.75

were calculated

from

the data

in Table

3.

STOCCO, BEERS, AND WARNER

Nucleotide

crease while AMP increases. Moreover, it was observed that embryos from batch 1 (1967) depleted their stores of ADP and ATP within 5 hr under anoxia, whereas embryos from batch 2 (1969) at a similar stage of development. required several days to deplete these nucleotides (compare Fig. 1 and Table 6). Similar differences were noted in the rate of recovery of these nucleotides upon termination of anoxia. During the aerobic “recovery phase” we found that the concentrations of the adenosine nucleotides in embryos from batch 1 were fully restored within 15 min in air, whereas several hours in air were required to restore the levels of these nucleotides in embryos from batch 2 (compare Fig. 1 and Table 4). At the present time we are unable to offer an explanation for the differences observed in the metabolic rates of the adenosine nucleotides in embryos from batches 1 and 2. According to Dutrieu and ChristiaBlanchine (1966) Artemiu embryos are able to withstand at least 5 months of anoxia with no subsequent effects upon the rate of development in air or upon the time of hatching. In contrast to these findings we observed that anoxia produces a delay in the resumption of development in embryos from batch 2, but that once morphogenesis resumes the rate of production of both prenauplii and nauplii is similar to controls exposed to air only. In support of these findings it should be noted that during the aerobic recovery period, the levels of all nucleotides, except for the diguanosine nucleotides, return to “normal” either just prior to, or coincident with, the resumption of morphogensis (see Fig. 2 and Table 4). Unfortunately, embryos from batch 1 were subjected to anoxia for only short periods of time (24 hrs) and are not directly comparable; nevertheless, no delay in resumption of development was observed in these embryos after 24 hr in an N, atmosphere. From these studies it appears that the delay in resumption of development, if any, following periods of

Metabolism

in Artemia

Embryos

491

anoxia correlates well with the time required for recovery of the nucleotide levels. In an earlier study, Ewing and Clegg (1969) reported that nauplii, unlike early encysted embryos, are unable to regulate anaerobic carbohydrate metabolism and are senstitive to anoxia surviving only a few hours in an anaerobic environment. In the present study we observed that although the pattern of anaerobic adenosine nucleotide metabolism appears to be similar at all developmental stages examined, the pattern of anaerobic guanosine nucleotide metabolism differs. When nauplii are maintained anaerobically for up to 5 hr (longer periods of anoxia were found to be lethal) the expected utilization pattern (compared to younger embryos) of GDP, GTP, and Gp,G does not occur. Moreover, the level of Gp,G actually increases in response to anoxia in nauplii. From these observations it appears that as the embryos undergo the transition from encysted forms to swimming nauplii, control of guanosine nucleotide metabolism is altered. It should also be emphasized that all anaerobic experiments were limited to conditions tihere the effects of anoxia are completely reversible. It is not uncommon to find that only 50% of commercially available Artemia cysts are viable. To eliminate the possibility that the changes occuring anaerobically are due only to the nonviable embryos in our preparations, we separated encysted embryos that failed to hatch over a 4-day period at 30°C and subjected them to anoxia for 7 days. Although the nucleotide levels in the nonviable embryos differed in some respects to the total population, it was evident that little, if any, anaerobic metabolism occurs in the nonviable embryos. Therefore, we concluded that the changes in nucleotide metabolism in response to anoxia are true reflections of the response of viable embryos to the stressesof anoxia. The stability of Artemia embryos to the

492

DEVELOPMENTAL

BIOLOGY

stresses of anoxia is of great adaptive significance and allows for completion of development when the environmental conditions become favorable. Previous studies have indicated that mitosis, and therefore embryonic development, is impossible in the absence of oxygen leading to irreversible damage and death of the embryos (see Needham, 1931). However, it has been shown that both frog and trout embryos complete cleavage but do not gastrulate in the absence of oxygen (Brachet, 1934; Devillers and Rosenberg, 1953). In these embryos the ADP/ATP levels may be sufficient to permit completion of cleavage, whereas the more complex events associated with gastrulation are unable to function properly in the absence of an efficient energy-generating system. The ability of Artemia embryos to undergo morphogenesis in the absence of cell division and DNA synthesis has been clearly demonstrated (Nakanishi et al., 1962; Warner and McClean, 1968). These findings may account for the fact that the deleterious effects normally associated with anoxia are not manifested in these embryos until just prior to hatching when the embryos resume DNA synthesis and cell division. Finally, it should be noted that the large amount of diguanosine tetraphosphate in these embryos may provide sufficient phosphate-bond energy to maintain the cellular and developmental integrity of arrested embryos for several months or until an adequate amount of oxygen is restored to the environment. We Holmes

extend our appreciation to Mrs. for her excellent technical assistance.

Jane

A.

REFERENCES BARTH, L., and JAEGER, L. J. (1947). Phosphorylation in the frog’s egg. Physiol. Zoo/. 20, 135-146. BRACHET, J. (1934). Etude du metabolism de I’oeuf de Grenouille (Ranu fusca) au tours du dbveloppement. 1. La respiration et la glycolyse, de la segmentation 21l’&closion. Arch. Viol. 45,611-727. BRACHET, J., and LEDOUX, L. (1955). The action of

VOLUME

21, 1972

ribonuclease on the division of amphibian eggs. Exp. Cell Res., Suppl. 3, 27-39. CLEGG, J. S., WARNER, A. H., and FINAMORE, F. J. (1967). Evidence for the function of P’, P”-diguanosine 5’.tetraphosphate in the development of Artemia salina. J. Biol. Chem. 242, 1938-1943. COHN, W. E. (1950). The anion-exchange separation of ribonucleotides. J. Amer. Chem. Sot. 72, 14711478. COHN, W. E. (1955). The separation of nucleic acid derivatives by chromatography on ion-exchange columns. In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, p. 218. Academic Press, New York. DEVILLERS, C., and ROSENBERG, J. (1953). Les premieres phases du dtveloppement de l’oeuf de S&no irredeus en anaerobiose. C. R. Acad. Sci. 237, 1561-1562. DUTRIEU, J., and CHRISTIA-BLANCHINE, D. (1966). RBsistance des oeufs durables hydrates d’drtemia salina B l’anoxia. C. R. Acad. Sci. Ser. D. 263,9981000. EWING, R. (1968). An analysis of lactate dehydrogenase and anaerobiosis in Artemia salina. Doctoral Dissertation, University of Miami. EWING, R. D., and CLEGG, J. S. (1969). Lactic dehydrogenase activity and anaerobic metabolism during embryonic development in Artemia salina. Comp. Biochem. Hzysiol. 31, 297-307. FINAMORE, F. J., and CLEGG, J. S. (1969). Biochemical aspects of morphogenesis in the brine shrimp Artemia salina. In “The Cell Cycle. Gene-Enzyme Interactions” (G. M. Padilla, G. L. Whitson, and eds.), pp. 249-278. Academic I. L. Cameron, P&s, New York. FINAMORE, F. J., and WARNER, A. H. (1963). The occurrence of P’, P’-diguanosine 5’-tetraphosphate in brine shrimp eggs. J. Biol. Chem. 238, 344-348. FINAMORE, F. J., and WARNER , A. H. (1966). P’,P’diguanosine 5’.tetraphosphate from brine shrimp eggs. In “Biochemical Preparations” (A. C. Maehly, ed.), Vol. 11, pp. 27-30. Wiley, New York. ISHIHARA, N., and KIKUCHI, G. (1968). Studies on the functional relationships between the phosphopyruvate synthesis and the substrate level phosphorylation in guinea-pig liver mitochondria. Biochim. Biophys. Acta 153, 733-748. MCCLEAN, D. K., and WARNER, A. H. (1971). Aspects of nucleic acid metabolism during development of the brine shrimp Artemia salina. Develop. Biol. 24, 88-105. NAKANISHI, Y. H., IWASAKI, T., OKIGAKI, T., and KATO, H. (1962). Cytological studies of Artemia salina. I. Embryonic development without cell multiplication after the blastula stage in encysted dry eggs. Annot. Zool. Jap. 35, 223-228.

STOCCO,

BEERS,

AND

WARNER

Nucleotide

J. (1931). Anaerobiosis in embryonic life. In “Chemical Embryology” Vol. 2, pp. 7422746. Cambridge Univ. Press, London and New York. SAZ, H. J., and LESCURE, 0. L. (1966). Interrelationships between carbohydrate and lipid metabolism of Ascaris lumbricoides egg and adult stages. Camp. Biochem. Physiol. 18, 845-857. VOLKIN, E., and COHN, W. E. (1954). Estimation of nucleic acids. Methods Biochem. Anal. 1, 287-303. VON BRAND, T. (1946). Anaerobiosis in invertebrates. In “Biodynamics” Monograph No. 4. Biodynamics, Normandy, Missouri. NEEDHAM,

Metabolism

in Artemia

Embryos

493

A. H., and FINAMORE, F. J. (1965). Isolation, purification, and characterization of P’, P”diguanosine 5’.tetraphosphate asymmetricalpyrophosphohydrolase from brine shrimp eggs. Biochemistry 4, 15681575. WARNER, A. H., and FINAMORE, F. J. (1967). Nucleotide metabolism during brine shrimp embryogenesis. J. Biol. Chem. 242, 1933-1937. WARNER, A. H., and MCLEAN, D. K. (1968). Studies on the biosynthesis and role of diguanosine tetraphosphate during growth and development of Artemia salina. Deuelop. BioE. 18,27%293.

WARNER,