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Lactate dehydrogenase activity and anaerobic metabolism during embryonic development in Artemia salina
Comp. Biochem. Physiol,, 1969, Vol. 31, pp. 297 to 307. Pergamon Press. Printed in Great Britain
LACTATE DEHYDROGENASE ACTIVITY AND ANAEROBIC METABOLISM DURING EMBRYONIC DEVELOPMENT IN A R T E M I A S A L I N A R. D. E W I N G *
and J. S. C L E G G
Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables, Florida 33124 (Received 24 March 1969)
A b s t r a c t - - 1 . When incubated aerobically, encysted gastrulae of the brine shrimp, Artemia salina, utilize trehalose and synthesize glycogen and glycerol during pre-emergence development; anaerobiosis prevents those changes and does not result in the accumulation of lactic acid. 2. Under aerobic conditions newly hatched nauplii (larvae) appear to conserve glycogen by utilizing glycerol. When made anaerobic they utilize glycogen, not glycerol, and accumulate large concentrations of lactic acid. 3. Under anaerobic conditions the nauplii lose motility within the first 2 hr and die in less than 12 hr. In contrast, encysted embryos are not killed by prolonged anaerobiosis. 4. Lactic acid dehydrogenase (LDH) activity remains constant at a low level during pre-emergence development but increases markedly during emergence and hatching. 5. This increase in L D H activity is correlated with the onset of lactic acid production in response to anaerobiosis and with the emergence of the embryos and their subsequent transformation into nauplii.
INTRODUCTION IN 1966 Dutrieu & Chrestia-Blanchine made the remarkable observation that hydrated encysted gastrulae of the brine shrimp, A r t e m i a salina, can tolerate exposure to 100% N 2 for at least 5 months, during which time they apparently do not utilize their lipid and carbohydrate reserves to arty appreciable extent. F u r t h e r more, these gastrulae, when returned to aerobic conditions, produced swimming nauplii (larvae) at the same rate and percentage as gastrulae not incubated anaerobically, indicating that their development was brought to a standstill b y anaerobiosis, but in a way that was completely reversible. I n an attempt to understand the basis of this unusual response, we have analyzed these embryos for the enzyme lactate dehydrogenase ( L D H ) and have examined their response to anaerobiosis as they undergo development into larvae. * Present address: Biology Division, P.O. Box Y, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831. 297
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M A T E R I A L S AND M E T H O D S Most of the experiments utilized brine shrimp cysts (encysted dried gastrulae) of the San Francisco variety, purchased in 1964 from Brine Shrimp Sales Co., Hayward, California. In a few instances embryos from the Great Salt Lake (Utah), obtained in 1967 from Longlife Fishfood Products, Harrison, N.J., were used. These two varieties are quite similar with regard to their response to anaerobiosis. Before use a large population of embryos was washed and redesiccated as described previously (Clegg, 1964). The washed embryos were stored at room temperature in a desiccator over anhydrous granular CaC1,.
Incubation procedures Washed embryos were prepared for incubation by hydrating and sterilizing them for 3 hr in 7% antiformin solution at 0°C (Nakanishi et al., 1962). The embryos were washed thoroughly with 600-1000 ml of sterile 0.2 M NaC1, rinsed with distilled water, blotted, weighed, and placed in flasks for incubation at 30°C in sterile sea water. In the case of aerobic incubations the flasks were continously agitated to ensure an adequate oxygen supply. Nauplii were obtained by incubating embryos in Petri dishes containing Milliporefiltered sea water at 30°C for 24 hr under sterile conditions. The nauplii were freed from shells, dead nauplii and unhatched embryos by their phototactic movements toward a light source. For each experiment the percentage of embryos emerged and hatched from a sample of the original population was scored at intervals by the criteria of Jennings & Whitaker (1941), with the final percentages of emergence and hatching determined after 48 hr. "Emergence" refers to the onset of escape of the pre-nauplius from the cyst; "hatching" refers to the escape of the swimming nauplius from an enclosing envelope. "Pre-emergence development" is that period of development occurring between hydration of the dried gastrula and emergence of the partially formed nauplius (pre-nauplius) from the cyst. Incubation under nitrogen was as described by Gregg (1962): a flow of nitrogen was continuously bubbled through the flasks. The nitrogen was first hydrated by passing it through distilled water to prevent evaporation of the incubation medium during the experiment. In initial experiments the nitrogen was purified by passing it through a series of flasks containing solutions known to absorb specific gases: 40% K O H for absorbing CO,, SO,, SOs, HsS, and C1,; 2-5% pyrogallol in 40% K O H for absorbing oxygen; 0.1 M CuCI, in 40% K O H for absorbing carbon monoxide and acetylene; distilled water to trap any watersoluble material and to hydrate the nitrogen before it passed into the incubation flasks. Since no significant differences in results were obtained using this elaborate washing procedure, it was not employed in most of the experiments to be presented here. After the desired time of incubation, the flasks were plunged into ice and the embryos or nauplii washed thoroughly with distilled water on cloth filter supports and transferred to T e n Broeck glass homogenizers. Chemical analysis The embryos were homogenized in 5% T C A and the supernatant and precipitate separated by centrifugation. Aliquots of the supernatant were used for the following chemical assays. Lactic acid was measured by the method of Barker (1957). Corrections for reaction with trehalose and glycerol were made when applicable. Trehalose and glycerol were isolated by continuous ascending paper chromatography using an n-butanol : 95% ethanol : acetone : water (5 : 4 : 3 : 2 v/v) mixture as solvent. The trehalose fraction was eluted with water, lyophilized, redissolved in a known volume of water and assayed by the phenol method of Dubois et al. (1956). Glycerol was measured by the method of Burton (1957).
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Glycogen was isolated from the embryos and nauplii as described previously (Clegg, 1964) and assayed by the anthrone method (Dimler et al., 1952). Protein was estimated by the method of Lowry et al. (1951).
Assay of lactate dehydrogenase activity (LDH) The embryos or nauplii were homogenized in Ten Broeck homogenizers with 0.05 M sodium phosphate buffer, pH 7'0. The extract was then centrifuged at 10,000 g for 30 min at 4°C. Carotenoids and other lipids were aspirated from the top of the extract and the opalescent supematant carefully removed and used as a source of enzyme. LDH activity was measured spectrophotometrically by the disappearance of NADH~ absorbance at 340 m/z in a Beckman DB spectrophotometer equipped with a potentiometric recorder. Temperature of the assay, 30 + I°C. The standard reaction mixture contained 0"1 ml enzyme preparation, 0.1 ml 0"01 M sodium pyruvate and 0"2 ml 0"6 mM NADt-I 2 brought to a final volume of 3"0 ml with 0"05 M sodium phosphate buffer, pH 7"0. Initial rates were measured and expressed in units per mg protein, a unit of activity being the oxidation of 1"0/zmole of NADH2/min. Protein was estimated by the method of Lowry et al. (1951) using a washed 5% trichloroacetic acid precipitate of the enzyme preparation dissolved in 0.5 M NaOH. RESULTS Metabolic response of encysted gastrulae to aerobic and anaerobic conditions When incubated aerobically, the gastrulae utilized trehalose, synthesized glycogen and, to a lesser extent, glycerol (Fig. 1, closed circles) as expected from previous studies (Dutrieu, 1960; Clegg, 1962, 1964). However, when gastrulae were incubated anaerobically, no appreciable changes in the concentrations of these compounds occurred during the 8-hr period examined (Fig. 1, open circles). Under aerobic and anaerobic conditions the low concentration of lactic acid present in the dormant gastrula remained the same and no detectable lactic acid was present in the incubation medium. When gastrulae that had been incubated anaerobically for 8 hr were returned to air they produced nauplii in the same percentage and at the same rate as gastrulae not exposed to anaerobiosis (about 72 per cent). These results support those of Dutrieu & Chrestia-Blanchine (1966) and indicate that an anaerobic metabolism based on the utilization of carbohydrate and accumulation of lactic acid does not occur in gastrulae at a rate detectable within 8 hr of incubation.
Metabolic response of swimming nauplii to aerobic and anaerobic conditions As the gastrula develops into a nauplius essentially all of the trehalose is utilized and glycogen, which is synthesized during this same period, becomes the major stored carbohydrate of the nauplius (Dutrieu, 1960; Clegg, 1962, 1964). Figure 2 shows, however, that nauplii incubated aerobically (closed circles) do not immediately utilize appreciable amounts of glycogen but do metabolize their glycerol. T h e concentration of lactic acid in aerobic nauplii is negligible. In contrast, nauplii incubated anaerobically (open circles in Fig. 2) do not utilize glycerol but extensively utilize glycogen with a concomitant increase in
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lactic acid. Such data clearly illustrate the striking difference between the response of gastrulae and nauplii to anaerobic conditions. 400
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FIG. 1. Metabolic response of encysted gastrulae to aerobic (--@--) and anaerobic (--©--) incubation. The gastrulae were hydrated at 0°C for 3 hr then incubated for the times designated at 30°C in sea water. The concentrations of lactic acid in Fig. 2 represent the sum of lactic acid present in the nauplius and that released into the medium during incubation. A breakdown of these, given in Table 1, reveals that about 90 per cent of the total lactic acid is present in the nauplii, the remainder being found in the medium. Table 2 compares the decrease in glycogen concentration with the increase in lactic acid concentration during anaerobiosis in nauplii. Inspection of these data reveals that most of the glycogen utilized can be accounted for by the observed increase in lactic acid.
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FIO. 2. Metabolic response of nauplii to aerobic ( - - O - - ) and anaerobic ( - - O - - ) incubation. Nauplii that had hatched within 3 hr were incubated at 30°C in sea water. Since the nauplius contains less protein than the encysted gastrula these values cannot be compared directly with those in Fig. 1. TABLE
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ANAEROBIC
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Lactic acid (/zg/mg nauplius protein) Hours incubation*
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1.9 23.6 40.2 64.5
< 2.4 26.2 44.5 74.5
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301
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R. D. EWlNG AND J. S. CLEGG TABLE 2--COMPARISONOF GLYCOGENUTILIZATION AND LACTIC ACID PRODUCTION IN NAUPLII DURING ANAEROBIOSIS
/~g/mg Nauplius protein Hours incubation*
Glycogen decrease
Lactic acid increase
Difference
0-0.5 0"5-1"0 1'0-2"0 0-2"0
22.4 20"7 38"6 81 "8
25"1 18"3 30.1 74.5
2-7 2"4 8"5 7"3
* Incubation conditions same as described in Fig. 2. Table 3 summarizes the effects of anaerobiosis on motility and viability of nauplii. Compared with controls (0 hr anaerobiosis), no appreciable decrease in viability was observed during the first 2 hr of anaerobiosis although all of the nauplii were immobilized. T h e nauplii began to die after 2-4 hr of anaerobiosis and all were dead after 12 hr. These data on nanplii are in striking contrast to those on encysted gastrulae, which survived 5 months of anaerobiosis with no decrease in viability (Dutrieu & Chrestia-Blanchine, 1966). TABLE 3--MOTILITY AND VIABILITY OF NAUPLII DURING ANAEROBIOSIS
Hours under N2
Motility
Percentage recovery*
0 0"5 1"0 2'0 4"0 12"0
Normal Reduced Much reduced Not motile Not motile Not motile
91 88 93 84 61 0
* Represents percentage of nauplii swimming normally and molted 48 hr after being returned to aerobic conditions. At least 200 nauplii examined for each incubation period.
Lactic acid dehydrogenase activity in extracts of gastrulae and nauplii Since the enzyme lactic acid dehydrogenase ( L D H ) plays a central role in the anaerobic metabolism of carbohydrate in most organisms we examined this enzyme activity in Artemia embryos.
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Table 4 shows that very low levels of L D H are detected in encysted gastrulae hydrated at 0°C. Extracts of nauplii, however, contain 35-40 times more L D H activity than those of gastrulae. This enzyme activity has been characterized in some detail and the results will be published elsewhere. Suffice it to say here that our enzyme assays were carried out at near optimal pH and concentrations of NADH 2 and pyruvate, and that mixing of gastrulae and nauplii extracts did not indicate the presence of an activator or inhibitor. T A B L E 4 - I - L A C T I C ACID DEHYDROGENASE ACTIVITY IN ENCYSTED GASTRULA AND NAUPLIUS EXTRACTS
L D H activity*
Encysted gastrula Nauplius Nauplius/gastrula
U n i t s t / m g protein
U n i t s t / m g wet wt.
0"014 0"570 41
3 x 10 -4 106 x 10 -4 35
* The 14,000 g supematant was used as the source of enzyme activity. t A/zmoles NADH2/min.
LDH activity and lactic acid production during development Having shown that gastrulae do not accumulate lactic acid during anaerobiosis but that nauplii do, and that the L D H activity detectable in extracts of nauplii is 35- to 40-fold higher than in gastrulae extracts, we carried out experiments to determine when these changes occur during development. The gastrulae were incubated aerobically for various periods in sea water at 30°C. At different times three samples of embryos were removed: one was analyzed for lactic acid, another was incubated under N~ for an additional hour and then analyzed for lactic acid and the third sample was analyzed for L D H activity. The percentages of emerged embryos and nauplii in these populations were also determined as described in Materials and Methods. The results are plotted in Fig. 3. The embryos begin to emerge after about 8-10 hr of incubation, and nauplii appear after 12-14 hr. The activity of L D H remains constant until about the onset of emergence and the embryos do not accumulate lactic acid in response to anaerobiosis during this same period of pre-emergence development. Once emergence begins in the population, however, the activity of L D H increases with time and the embryos exhibit a greater tendency to accumulate lactic acid during the 1-hr period of anaerobiosis. Thus, the onset of the ability to synthesize lactic acid in response to anaerobiosis is correlated with an increase in LDH activity, both of these events occurring when the embryo emerges from its shell and develops into a swimming nauplius.
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FIG. 3. Relationship between lactic acid dehydrogenase (LDH) activity and the ability to produce lactic acid in response to anaerobiosis during development. LDH activity (--Q--) is represented as units/mg protein; A lactic acid is the change in lactic acid concentration during 1 hr of anaerobiosis ( - - i F - ) ; percentage emergence (-- O--) ; percentage hatching (-- [3--). Vertical bars represent _+ the standard error. DISCUSSION Based on chemical studies (Dutrieu, 1960; Clegg, 1964) and R.Q. measurements (Dutrieu, 1960; Muramatsu, 1960; Emerson, 1963 ; Clegg, 1964), it is quite clear that trehalose is the major and perhaps the exclusive substrate for aerobic energy metabolism during the pre-emergence development of encysted Artemia embryos. When incubated anaerobically, however, these embryos do not utilize trehalose (Fig. 1). In fact, Dutrieu & Chrestia-Blanchine (1966) observed a very small increase in the concentration of this sugar during prolonged anaerobiosis (2 months) along with a slight decrease in glycogen and slight increases in glycerol and total "lipids". Consequently, we conclude that the utilization of trehalose in these embryos necessitates the presence of molecular oxygen. Furthermore, our results (Fig. 1) and especially those of Dutrieu & Chrestia-Blanchine (1966) suggest that the rate of carbohydrate metabolism is reduced almost to a standstill by anaerobic conditions. It would therefore seem that these embryos respond to oxygen lack by reducing their metabolic activity, unlike the vast majority of organisms which show increased
LACTATE DEHYDROGENASE ACTIVITY AND ANAEROBIC METABOLISM I N ARTEMIA SALINA
30~
carbohydrate utilization with concurrent accumulation of large amounts of lactic acid or other metabolic end products and eventual death of the organism if anaerobiosis is prolonged. We agree with Dutrieu & Chrestia-Blanchine (1966) that this reduction in metabolic activity might provide a basis for the tolerance of the gastrula to anaerobiosis. But an interesting implication of this suggestion is that the embryo, fully hydrated and at temperatures exceeding 20°C, can maintain its integrity over periods exceeding 5 months with a very low rate of energy metabolism. The alternative possibilities--that they possess a unique type of anaerobic metabolism not based on the utilization of known carbohydrates or that they have some other source of free energy--are equally interesting. Since it has been shown that the level of ATP in encysted gastrulae is extremely low (Warner & Finamore, 1967), a supply of free energy in this form is not available. However, Artemia embryos do contain very large amounts of p1, p4_diguanosine_5, tetraphosphate (Finamore & Warner, 1963). Although this nucleotide is not utilized to any appreciable extent during pre-emergence development under aerobic conditions (Warner & Finamore, 1967), it remains to be seen whether or not it is metabolized anaerobically. The response of the nauplius to anaerobic conditions contrasts sharply with that of the encysted embryo: the nauplius rapidly accumulates large amounts of lactic acid (Table 1, Fig. 2)--probably at the expense of glycogen (Table 2, Fig. 2)-loses motility and dies at 4-12 hr of anaerobiosis (Table 3). Aerobically, the newly hatched nauplius appears to utilize glycerol rather than glycogen (Fig. 2). The extent to which glycerol serves as a substrate for energy metabolism has not yet been evaluated but it is known that this substance is metabolized within the nauplius and is not released into the medium (Clegg, 1962). The level of L D H activity that we have detected in the encysted gastrula is extremely low (Table 4) and remains so throughout pre-emergence development (Fig. 3). During this same period the embryo does not degrade carbohydrate (Dutrieu & Chrestia-Blanchine, 1966) nor accumulate lactate under anaerobic conditions (Fig. 3). Thus we conclude that the L D H activity we detected in extracts is either insufficient to support anaerobic glycolysis or is in some way inactivated or separated from its substrate within the intact embryo. In view of this, and the observed correlation between the increase in L D H activity, the onset of lactic acid formation in response to anaerobiosis, and the emergence and hatching processes (Fig. 3), it is possible that the synthesis (or activation) of L D H may be a means of regulating carbohydrate metabolism during development. We should point out that several other metabolic and developmental changes are known to occur at about the time of emergence: (a) cell division, absent throughout pre-emergence development, is resumed at emergence (Nakanishi et al., 1962, 1963) and the same is probably true for DNA synthesis (Bellini, 1960); (b) p1,p4_ diguanosine-5' tetraphosphate (diGDP) which is present in very large amounts in encysted gastrulae (Finamore & Warner, 1963) and remains reasonably constant throughout pre-emergenee development (Warner & Finamore, 1967), is extensively utilized after emergence and hatching have occurred (Warner & Finamore, 1967;
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R. D. E W I N G AND J. S. CLEGG
Clegg et al., 1967; W a r n e r & McClean, 1968). T h e metabolism of d i G D P after hatching m a y be related to the resumption of D N A synthesis (Finamore & Clegg, 1969); (c) several enzyme activities that remain constant during pre-emergence development show abrupt increases at about the time of emergence (Bellini, 1957a, b; Bellini & Lavizzari, 1958 ; Urbani & Bellini, 1958; Russo-Caia & Bellini, 1960) in m u c h the same fashion that we have observed for L D H (Fig. 3). T h e s e studies make clear the fact that major transitions in metabolism accompany the development of emerged embryos into larvae, and indicate that these embryos are well suited to the study of metabolic regulation during embryonic development. Acknowledgements--This study was supported by research grants from USPHS (HD03478) and from NASA (NGR 10-007-010). One of the authors (R. D. E.) held a USPHS pre-doctoral fellowship during the period of research reported. The helpful criticisms and assistance of M. Niblock in the preparation of the manuscript and the technical assistance of G. Gutten are gratefully acknowledged.
REFERENCES BARKER S. B. (1957) Preparation and colorimetric determination of lactic acid. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 3, pp. 241-246. Academic Press, New York. BELLINI L. (1957a) Studio delle proteinasi e dipeptidasi nelle sviluppo di Artemia salina. A t t i Aeead. nazion. Lincei, Rend. Set. 8, 22, 340-345. BELLINI L. (1957b) Studio delle amilasi nello sviluppo di Artemia salina Leach. Atti Accad. nazion. Lincei, Rend. Ser. 8, 23, 303-307. BELLINI L. (1960) Osservazioni sugli acidi nucleici nello sviluppo di Artemia salina Leach. La Ricerea Scientifica 30, 816-822. BELLINI L. & LAVIZZARIG. S. (1958) Studio delle lipase nello sviluppo di Artemia salina Leach. A t t i Accad. nazion. Lincei, Rend. Set. 8, 24, 92-95. BURTON R. M. (1957) The determination of glycerol and dihydroxyacetone. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O. ), Vol. 3, pp. 246-249. Academic Press, New York. CLEGG J. S. (1962) Free glycerol in dormant cysts of the brine shrimp Artemia salina, and its disappearance during development. Biol. Bull. mar. biol. Lab., Woods Hole 123, 295-301. CLEGG J. S. (1964) The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina, ft. exp. Biol. 41, 879892. CLEGG J. S., WARNER A. H. & FINAMOREF. J. (1967) Evidence for the function of p1,p4_ diguanosine 5'-tetraphosphate in the development of Artemia salina, ft. biol. Chem. 242, 1938-1943. DIMLER R. J., SCHAEYERW. C., WISE C. S. & RIST C. E. (1952) Quantitative paper chromatography of n-glucose and its oligosaccharides. Analyt. Chem. 24, 1411-1414. DUBOIS M., GILLES K. A., HAMILTONJ. K., RIBERSP. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356. DUTmEU J. (1960) Observations biochimiques et physiologiques sur le d6veloppement d'Artemia salina Leach. Archs Zool. exp. gdn. 99, 1-133. DUTRIEU J. & CHRESTIA-BLANCHINED. (1966) R6sistance des oeufs durables hydrat6s d'Artemia salina ~t l'anoxie. C. r. Acad. Sci. Paris S6rie D. 263, 998-1000.
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EMERSON D. N. (1963) The metabolism of hatching embryos of the brine shrimp Artemia salina. Proc. S. Dakota Acad. Sci. 42, 131-135. FINAMORE F. J. & WA~ER A. H. (1963) The occurrence of P1,P*-diguanosine-5'-tetraphosphate in brine shrimp eggs. ~. biol. Chem. 238, 344-348. FINAMOREF. J. & CLEGGJ. S. (1969) Biochemical aspects of morphogenesis in the brine shrimp, Artemia salina. In The Cell Cycle. Gem-Enzyme Interactions (Edited by PADILLAG. M., WHITSON G. L. & CAMERON I. L.), pp. 249-278. Academic Press, New York. GREGG J. R. (1962) Anaerobic glycolysis in amphibian development. Biol. Bull. mar. biol. Lab., Woods Hole 123, 555-561. JENNINGSR. H. & WHITAKERn . M. (1941) The effect of salinity upon the rate of excystment of Artemia salina. Biol. Bull. mar. biol. Lab., Woods Hole 80, 194-201. LOWRY O. H . , ROSEBOROUGH INT. J., FARR A . L . & RANDALL R. J. (1951) P r o t e i n m e a s u r e m e n t with the Folin phenol reagent. 3¢. biol. Chem. 193, 265-275. NIURAMATSUS. (1960) Studies on the physiology of Artemia embryos--I. Respiration and its main substrate during early development. Embryologia 5, 95-106. NAKANISHIY. H., IWASAKIT., OKICAKIT. & KATOH. (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. NAKANISHIY. H., OKIOAKIT., KATOH. & IWASAKIT. (1963) Cytological studies of Artemia salina--II. Deoxyribonucleic acid content and the chromosomes in encysted dry eggs and nauplii. Proc. Japan Acad. 39, 306-309. RUSSO-CAIAS. & BELLINIL. (1960) Idrolisi enzimatica dell ATP nello sviluppo di ~lrtemia salina Leach diploide e tetraploide. 1st. Sci. Univ. Camerino, Rend. 1, 136-144. URBANI E. & BELLINI L. (1958) Studio delle ribonucleasi acida nella sviluppo di ~Irtemia salina Leach. Atti Accad. nazion. Lincei, Rend. 25, 198-201. WARNER A. H. & FINAMORE F. J. (1967) Nucleotide metabolism during brine shrimp embryogenesis, aT. biol. Chem. 242, 1933-1937. WARNERA. H. & MCCLEAYD. K. (1968) Studies on the biosynthesis and role of diguanosine tetraphosphate during the growth and development of Artemia salina. Devel. Biol. 18, 278-293. Key Word Index--Anoxia; anaerobiosis; lactic acid dehydrogenase; .xlrtemia salina; brine shrimp; development; carbohydrate metabolism during development; glycogen; trehalose; glycerol.
LACTATE DEHYDROGENASE ACTIVITY AND ANAEROBIC METABOLISM DURING EMBRYONIC DEVELOPMENT IN A R T E M I A S A L I N A R. D. E W I N G *
and J. S. C L E G G
Laboratory for Quantitative Biology, Department of Biology, University of Miami, Coral Gables, Florida 33124 (Received 24 March 1969)
A b s t r a c t - - 1 . When incubated aerobically, encysted gastrulae of the brine shrimp, Artemia salina, utilize trehalose and synthesize glycogen and glycerol during pre-emergence development; anaerobiosis prevents those changes and does not result in the accumulation of lactic acid. 2. Under aerobic conditions newly hatched nauplii (larvae) appear to conserve glycogen by utilizing glycerol. When made anaerobic they utilize glycogen, not glycerol, and accumulate large concentrations of lactic acid. 3. Under anaerobic conditions the nauplii lose motility within the first 2 hr and die in less than 12 hr. In contrast, encysted embryos are not killed by prolonged anaerobiosis. 4. Lactic acid dehydrogenase (LDH) activity remains constant at a low level during pre-emergence development but increases markedly during emergence and hatching. 5. This increase in L D H activity is correlated with the onset of lactic acid production in response to anaerobiosis and with the emergence of the embryos and their subsequent transformation into nauplii.
INTRODUCTION IN 1966 Dutrieu & Chrestia-Blanchine made the remarkable observation that hydrated encysted gastrulae of the brine shrimp, A r t e m i a salina, can tolerate exposure to 100% N 2 for at least 5 months, during which time they apparently do not utilize their lipid and carbohydrate reserves to arty appreciable extent. F u r t h e r more, these gastrulae, when returned to aerobic conditions, produced swimming nauplii (larvae) at the same rate and percentage as gastrulae not incubated anaerobically, indicating that their development was brought to a standstill b y anaerobiosis, but in a way that was completely reversible. I n an attempt to understand the basis of this unusual response, we have analyzed these embryos for the enzyme lactate dehydrogenase ( L D H ) and have examined their response to anaerobiosis as they undergo development into larvae. * Present address: Biology Division, P.O. Box Y, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831. 297
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R . D . EWlNG AND J. S. CLEGG
M A T E R I A L S AND M E T H O D S Most of the experiments utilized brine shrimp cysts (encysted dried gastrulae) of the San Francisco variety, purchased in 1964 from Brine Shrimp Sales Co., Hayward, California. In a few instances embryos from the Great Salt Lake (Utah), obtained in 1967 from Longlife Fishfood Products, Harrison, N.J., were used. These two varieties are quite similar with regard to their response to anaerobiosis. Before use a large population of embryos was washed and redesiccated as described previously (Clegg, 1964). The washed embryos were stored at room temperature in a desiccator over anhydrous granular CaC1,.
Incubation procedures Washed embryos were prepared for incubation by hydrating and sterilizing them for 3 hr in 7% antiformin solution at 0°C (Nakanishi et al., 1962). The embryos were washed thoroughly with 600-1000 ml of sterile 0.2 M NaC1, rinsed with distilled water, blotted, weighed, and placed in flasks for incubation at 30°C in sterile sea water. In the case of aerobic incubations the flasks were continously agitated to ensure an adequate oxygen supply. Nauplii were obtained by incubating embryos in Petri dishes containing Milliporefiltered sea water at 30°C for 24 hr under sterile conditions. The nauplii were freed from shells, dead nauplii and unhatched embryos by their phototactic movements toward a light source. For each experiment the percentage of embryos emerged and hatched from a sample of the original population was scored at intervals by the criteria of Jennings & Whitaker (1941), with the final percentages of emergence and hatching determined after 48 hr. "Emergence" refers to the onset of escape of the pre-nauplius from the cyst; "hatching" refers to the escape of the swimming nauplius from an enclosing envelope. "Pre-emergence development" is that period of development occurring between hydration of the dried gastrula and emergence of the partially formed nauplius (pre-nauplius) from the cyst. Incubation under nitrogen was as described by Gregg (1962): a flow of nitrogen was continuously bubbled through the flasks. The nitrogen was first hydrated by passing it through distilled water to prevent evaporation of the incubation medium during the experiment. In initial experiments the nitrogen was purified by passing it through a series of flasks containing solutions known to absorb specific gases: 40% K O H for absorbing CO,, SO,, SOs, HsS, and C1,; 2-5% pyrogallol in 40% K O H for absorbing oxygen; 0.1 M CuCI, in 40% K O H for absorbing carbon monoxide and acetylene; distilled water to trap any watersoluble material and to hydrate the nitrogen before it passed into the incubation flasks. Since no significant differences in results were obtained using this elaborate washing procedure, it was not employed in most of the experiments to be presented here. After the desired time of incubation, the flasks were plunged into ice and the embryos or nauplii washed thoroughly with distilled water on cloth filter supports and transferred to T e n Broeck glass homogenizers. Chemical analysis The embryos were homogenized in 5% T C A and the supernatant and precipitate separated by centrifugation. Aliquots of the supernatant were used for the following chemical assays. Lactic acid was measured by the method of Barker (1957). Corrections for reaction with trehalose and glycerol were made when applicable. Trehalose and glycerol were isolated by continuous ascending paper chromatography using an n-butanol : 95% ethanol : acetone : water (5 : 4 : 3 : 2 v/v) mixture as solvent. The trehalose fraction was eluted with water, lyophilized, redissolved in a known volume of water and assayed by the phenol method of Dubois et al. (1956). Glycerol was measured by the method of Burton (1957).
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Glycogen was isolated from the embryos and nauplii as described previously (Clegg, 1964) and assayed by the anthrone method (Dimler et al., 1952). Protein was estimated by the method of Lowry et al. (1951).
Assay of lactate dehydrogenase activity (LDH) The embryos or nauplii were homogenized in Ten Broeck homogenizers with 0.05 M sodium phosphate buffer, pH 7'0. The extract was then centrifuged at 10,000 g for 30 min at 4°C. Carotenoids and other lipids were aspirated from the top of the extract and the opalescent supematant carefully removed and used as a source of enzyme. LDH activity was measured spectrophotometrically by the disappearance of NADH~ absorbance at 340 m/z in a Beckman DB spectrophotometer equipped with a potentiometric recorder. Temperature of the assay, 30 + I°C. The standard reaction mixture contained 0"1 ml enzyme preparation, 0.1 ml 0"01 M sodium pyruvate and 0"2 ml 0"6 mM NADt-I 2 brought to a final volume of 3"0 ml with 0"05 M sodium phosphate buffer, pH 7"0. Initial rates were measured and expressed in units per mg protein, a unit of activity being the oxidation of 1"0/zmole of NADH2/min. Protein was estimated by the method of Lowry et al. (1951) using a washed 5% trichloroacetic acid precipitate of the enzyme preparation dissolved in 0.5 M NaOH. RESULTS Metabolic response of encysted gastrulae to aerobic and anaerobic conditions When incubated aerobically, the gastrulae utilized trehalose, synthesized glycogen and, to a lesser extent, glycerol (Fig. 1, closed circles) as expected from previous studies (Dutrieu, 1960; Clegg, 1962, 1964). However, when gastrulae were incubated anaerobically, no appreciable changes in the concentrations of these compounds occurred during the 8-hr period examined (Fig. 1, open circles). Under aerobic and anaerobic conditions the low concentration of lactic acid present in the dormant gastrula remained the same and no detectable lactic acid was present in the incubation medium. When gastrulae that had been incubated anaerobically for 8 hr were returned to air they produced nauplii in the same percentage and at the same rate as gastrulae not exposed to anaerobiosis (about 72 per cent). These results support those of Dutrieu & Chrestia-Blanchine (1966) and indicate that an anaerobic metabolism based on the utilization of carbohydrate and accumulation of lactic acid does not occur in gastrulae at a rate detectable within 8 hr of incubation.
Metabolic response of swimming nauplii to aerobic and anaerobic conditions As the gastrula develops into a nauplius essentially all of the trehalose is utilized and glycogen, which is synthesized during this same period, becomes the major stored carbohydrate of the nauplius (Dutrieu, 1960; Clegg, 1962, 1964). Figure 2 shows, however, that nauplii incubated aerobically (closed circles) do not immediately utilize appreciable amounts of glycogen but do metabolize their glycerol. T h e concentration of lactic acid in aerobic nauplii is negligible. In contrast, nauplii incubated anaerobically (open circles in Fig. 2) do not utilize glycerol but extensively utilize glycogen with a concomitant increase in
300
R. D. EWlNOANDJ. S. CLECG
lactic acid. Such data clearly illustrate the striking difference between the response of gastrulae and nauplii to anaerobic conditions. 400
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FIG. 1. Metabolic response of encysted gastrulae to aerobic (--@--) and anaerobic (--©--) incubation. The gastrulae were hydrated at 0°C for 3 hr then incubated for the times designated at 30°C in sea water. The concentrations of lactic acid in Fig. 2 represent the sum of lactic acid present in the nauplius and that released into the medium during incubation. A breakdown of these, given in Table 1, reveals that about 90 per cent of the total lactic acid is present in the nauplii, the remainder being found in the medium. Table 2 compares the decrease in glycogen concentration with the increase in lactic acid concentration during anaerobiosis in nauplii. Inspection of these data reveals that most of the glycogen utilized can be accounted for by the observed increase in lactic acid.
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FIO. 2. Metabolic response of nauplii to aerobic ( - - O - - ) and anaerobic ( - - O - - ) incubation. Nauplii that had hatched within 3 hr were incubated at 30°C in sea water. Since the nauplius contains less protein than the encysted gastrula these values cannot be compared directly with those in Fig. 1. TABLE
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ANAEROBIC
NAUPLII
Lactic acid (/zg/mg nauplius protein) Hours incubation*
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1.9 23.6 40.2 64.5
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R. D. EWlNG AND J. S. CLEGG TABLE 2--COMPARISONOF GLYCOGENUTILIZATION AND LACTIC ACID PRODUCTION IN NAUPLII DURING ANAEROBIOSIS
/~g/mg Nauplius protein Hours incubation*
Glycogen decrease
Lactic acid increase
Difference
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25"1 18"3 30.1 74.5
2-7 2"4 8"5 7"3
* Incubation conditions same as described in Fig. 2. Table 3 summarizes the effects of anaerobiosis on motility and viability of nauplii. Compared with controls (0 hr anaerobiosis), no appreciable decrease in viability was observed during the first 2 hr of anaerobiosis although all of the nauplii were immobilized. T h e nauplii began to die after 2-4 hr of anaerobiosis and all were dead after 12 hr. These data on nanplii are in striking contrast to those on encysted gastrulae, which survived 5 months of anaerobiosis with no decrease in viability (Dutrieu & Chrestia-Blanchine, 1966). TABLE 3--MOTILITY AND VIABILITY OF NAUPLII DURING ANAEROBIOSIS
Hours under N2
Motility
Percentage recovery*
0 0"5 1"0 2'0 4"0 12"0
Normal Reduced Much reduced Not motile Not motile Not motile
91 88 93 84 61 0
* Represents percentage of nauplii swimming normally and molted 48 hr after being returned to aerobic conditions. At least 200 nauplii examined for each incubation period.
Lactic acid dehydrogenase activity in extracts of gastrulae and nauplii Since the enzyme lactic acid dehydrogenase ( L D H ) plays a central role in the anaerobic metabolism of carbohydrate in most organisms we examined this enzyme activity in Artemia embryos.
LACTATE DEHYDROGENASE ACTIVITY AND ANAEROBIC METABOLISM IN A R T E M ! A S A L I N A
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Table 4 shows that very low levels of L D H are detected in encysted gastrulae hydrated at 0°C. Extracts of nauplii, however, contain 35-40 times more L D H activity than those of gastrulae. This enzyme activity has been characterized in some detail and the results will be published elsewhere. Suffice it to say here that our enzyme assays were carried out at near optimal pH and concentrations of NADH 2 and pyruvate, and that mixing of gastrulae and nauplii extracts did not indicate the presence of an activator or inhibitor. T A B L E 4 - I - L A C T I C ACID DEHYDROGENASE ACTIVITY IN ENCYSTED GASTRULA AND NAUPLIUS EXTRACTS
L D H activity*
Encysted gastrula Nauplius Nauplius/gastrula
U n i t s t / m g protein
U n i t s t / m g wet wt.
0"014 0"570 41
3 x 10 -4 106 x 10 -4 35
* The 14,000 g supematant was used as the source of enzyme activity. t A/zmoles NADH2/min.
LDH activity and lactic acid production during development Having shown that gastrulae do not accumulate lactic acid during anaerobiosis but that nauplii do, and that the L D H activity detectable in extracts of nauplii is 35- to 40-fold higher than in gastrulae extracts, we carried out experiments to determine when these changes occur during development. The gastrulae were incubated aerobically for various periods in sea water at 30°C. At different times three samples of embryos were removed: one was analyzed for lactic acid, another was incubated under N~ for an additional hour and then analyzed for lactic acid and the third sample was analyzed for L D H activity. The percentages of emerged embryos and nauplii in these populations were also determined as described in Materials and Methods. The results are plotted in Fig. 3. The embryos begin to emerge after about 8-10 hr of incubation, and nauplii appear after 12-14 hr. The activity of L D H remains constant until about the onset of emergence and the embryos do not accumulate lactic acid in response to anaerobiosis during this same period of pre-emergence development. Once emergence begins in the population, however, the activity of L D H increases with time and the embryos exhibit a greater tendency to accumulate lactic acid during the 1-hr period of anaerobiosis. Thus, the onset of the ability to synthesize lactic acid in response to anaerobiosis is correlated with an increase in LDH activity, both of these events occurring when the embryo emerges from its shell and develops into a swimming nauplius.
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R.D. EWlNGAND J. S. CLEGG
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FIG. 3. Relationship between lactic acid dehydrogenase (LDH) activity and the ability to produce lactic acid in response to anaerobiosis during development. LDH activity (--Q--) is represented as units/mg protein; A lactic acid is the change in lactic acid concentration during 1 hr of anaerobiosis ( - - i F - ) ; percentage emergence (-- O--) ; percentage hatching (-- [3--). Vertical bars represent _+ the standard error. DISCUSSION Based on chemical studies (Dutrieu, 1960; Clegg, 1964) and R.Q. measurements (Dutrieu, 1960; Muramatsu, 1960; Emerson, 1963 ; Clegg, 1964), it is quite clear that trehalose is the major and perhaps the exclusive substrate for aerobic energy metabolism during the pre-emergence development of encysted Artemia embryos. When incubated anaerobically, however, these embryos do not utilize trehalose (Fig. 1). In fact, Dutrieu & Chrestia-Blanchine (1966) observed a very small increase in the concentration of this sugar during prolonged anaerobiosis (2 months) along with a slight decrease in glycogen and slight increases in glycerol and total "lipids". Consequently, we conclude that the utilization of trehalose in these embryos necessitates the presence of molecular oxygen. Furthermore, our results (Fig. 1) and especially those of Dutrieu & Chrestia-Blanchine (1966) suggest that the rate of carbohydrate metabolism is reduced almost to a standstill by anaerobic conditions. It would therefore seem that these embryos respond to oxygen lack by reducing their metabolic activity, unlike the vast majority of organisms which show increased
LACTATE DEHYDROGENASE ACTIVITY AND ANAEROBIC METABOLISM I N ARTEMIA SALINA
30~
carbohydrate utilization with concurrent accumulation of large amounts of lactic acid or other metabolic end products and eventual death of the organism if anaerobiosis is prolonged. We agree with Dutrieu & Chrestia-Blanchine (1966) that this reduction in metabolic activity might provide a basis for the tolerance of the gastrula to anaerobiosis. But an interesting implication of this suggestion is that the embryo, fully hydrated and at temperatures exceeding 20°C, can maintain its integrity over periods exceeding 5 months with a very low rate of energy metabolism. The alternative possibilities--that they possess a unique type of anaerobic metabolism not based on the utilization of known carbohydrates or that they have some other source of free energy--are equally interesting. Since it has been shown that the level of ATP in encysted gastrulae is extremely low (Warner & Finamore, 1967), a supply of free energy in this form is not available. However, Artemia embryos do contain very large amounts of p1, p4_diguanosine_5, tetraphosphate (Finamore & Warner, 1963). Although this nucleotide is not utilized to any appreciable extent during pre-emergence development under aerobic conditions (Warner & Finamore, 1967), it remains to be seen whether or not it is metabolized anaerobically. The response of the nauplius to anaerobic conditions contrasts sharply with that of the encysted embryo: the nauplius rapidly accumulates large amounts of lactic acid (Table 1, Fig. 2)--probably at the expense of glycogen (Table 2, Fig. 2)-loses motility and dies at 4-12 hr of anaerobiosis (Table 3). Aerobically, the newly hatched nauplius appears to utilize glycerol rather than glycogen (Fig. 2). The extent to which glycerol serves as a substrate for energy metabolism has not yet been evaluated but it is known that this substance is metabolized within the nauplius and is not released into the medium (Clegg, 1962). The level of L D H activity that we have detected in the encysted gastrula is extremely low (Table 4) and remains so throughout pre-emergence development (Fig. 3). During this same period the embryo does not degrade carbohydrate (Dutrieu & Chrestia-Blanchine, 1966) nor accumulate lactate under anaerobic conditions (Fig. 3). Thus we conclude that the L D H activity we detected in extracts is either insufficient to support anaerobic glycolysis or is in some way inactivated or separated from its substrate within the intact embryo. In view of this, and the observed correlation between the increase in L D H activity, the onset of lactic acid formation in response to anaerobiosis, and the emergence and hatching processes (Fig. 3), it is possible that the synthesis (or activation) of L D H may be a means of regulating carbohydrate metabolism during development. We should point out that several other metabolic and developmental changes are known to occur at about the time of emergence: (a) cell division, absent throughout pre-emergence development, is resumed at emergence (Nakanishi et al., 1962, 1963) and the same is probably true for DNA synthesis (Bellini, 1960); (b) p1,p4_ diguanosine-5' tetraphosphate (diGDP) which is present in very large amounts in encysted gastrulae (Finamore & Warner, 1963) and remains reasonably constant throughout pre-emergenee development (Warner & Finamore, 1967), is extensively utilized after emergence and hatching have occurred (Warner & Finamore, 1967;
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R. D. E W I N G AND J. S. CLEGG
Clegg et al., 1967; W a r n e r & McClean, 1968). T h e metabolism of d i G D P after hatching m a y be related to the resumption of D N A synthesis (Finamore & Clegg, 1969); (c) several enzyme activities that remain constant during pre-emergence development show abrupt increases at about the time of emergence (Bellini, 1957a, b; Bellini & Lavizzari, 1958 ; Urbani & Bellini, 1958; Russo-Caia & Bellini, 1960) in m u c h the same fashion that we have observed for L D H (Fig. 3). T h e s e studies make clear the fact that major transitions in metabolism accompany the development of emerged embryos into larvae, and indicate that these embryos are well suited to the study of metabolic regulation during embryonic development. Acknowledgements--This study was supported by research grants from USPHS (HD03478) and from NASA (NGR 10-007-010). One of the authors (R. D. E.) held a USPHS pre-doctoral fellowship during the period of research reported. The helpful criticisms and assistance of M. Niblock in the preparation of the manuscript and the technical assistance of G. Gutten are gratefully acknowledged.
REFERENCES BARKER S. B. (1957) Preparation and colorimetric determination of lactic acid. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 3, pp. 241-246. Academic Press, New York. BELLINI L. (1957a) Studio delle proteinasi e dipeptidasi nelle sviluppo di Artemia salina. A t t i Aeead. nazion. Lincei, Rend. Set. 8, 22, 340-345. BELLINI L. (1957b) Studio delle amilasi nello sviluppo di Artemia salina Leach. Atti Accad. nazion. Lincei, Rend. Ser. 8, 23, 303-307. BELLINI L. (1960) Osservazioni sugli acidi nucleici nello sviluppo di Artemia salina Leach. La Ricerea Scientifica 30, 816-822. BELLINI L. & LAVIZZARIG. S. (1958) Studio delle lipase nello sviluppo di Artemia salina Leach. A t t i Accad. nazion. Lincei, Rend. Set. 8, 24, 92-95. BURTON R. M. (1957) The determination of glycerol and dihydroxyacetone. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O. ), Vol. 3, pp. 246-249. Academic Press, New York. CLEGG J. S. (1962) Free glycerol in dormant cysts of the brine shrimp Artemia salina, and its disappearance during development. Biol. Bull. mar. biol. Lab., Woods Hole 123, 295-301. CLEGG J. S. (1964) The control of emergence and metabolism by external osmotic pressure and the role of free glycerol in developing cysts of Artemia salina, ft. exp. Biol. 41, 879892. CLEGG J. S., WARNER A. H. & FINAMOREF. J. (1967) Evidence for the function of p1,p4_ diguanosine 5'-tetraphosphate in the development of Artemia salina, ft. biol. Chem. 242, 1938-1943. DIMLER R. J., SCHAEYERW. C., WISE C. S. & RIST C. E. (1952) Quantitative paper chromatography of n-glucose and its oligosaccharides. Analyt. Chem. 24, 1411-1414. DUBOIS M., GILLES K. A., HAMILTONJ. K., RIBERSP. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356. DUTmEU J. (1960) Observations biochimiques et physiologiques sur le d6veloppement d'Artemia salina Leach. Archs Zool. exp. gdn. 99, 1-133. DUTRIEU J. & CHRESTIA-BLANCHINED. (1966) R6sistance des oeufs durables hydrat6s d'Artemia salina ~t l'anoxie. C. r. Acad. Sci. Paris S6rie D. 263, 998-1000.
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EMERSON D. N. (1963) The metabolism of hatching embryos of the brine shrimp Artemia salina. Proc. S. Dakota Acad. Sci. 42, 131-135. FINAMORE F. J. & WA~ER A. H. (1963) The occurrence of P1,P*-diguanosine-5'-tetraphosphate in brine shrimp eggs. ~. biol. Chem. 238, 344-348. FINAMOREF. J. & CLEGGJ. S. (1969) Biochemical aspects of morphogenesis in the brine shrimp, Artemia salina. In The Cell Cycle. Gem-Enzyme Interactions (Edited by PADILLAG. M., WHITSON G. L. & CAMERON I. L.), pp. 249-278. Academic Press, New York. GREGG J. R. (1962) Anaerobic glycolysis in amphibian development. Biol. Bull. mar. biol. Lab., Woods Hole 123, 555-561. JENNINGSR. H. & WHITAKERn . M. (1941) The effect of salinity upon the rate of excystment of Artemia salina. Biol. Bull. mar. biol. Lab., Woods Hole 80, 194-201. LOWRY O. H . , ROSEBOROUGH INT. J., FARR A . L . & RANDALL R. J. (1951) P r o t e i n m e a s u r e m e n t with the Folin phenol reagent. 3¢. biol. Chem. 193, 265-275. NIURAMATSUS. (1960) Studies on the physiology of Artemia embryos--I. Respiration and its main substrate during early development. Embryologia 5, 95-106. NAKANISHIY. H., IWASAKIT., OKICAKIT. & KATOH. (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. NAKANISHIY. H., OKIOAKIT., KATOH. & IWASAKIT. (1963) Cytological studies of Artemia salina--II. Deoxyribonucleic acid content and the chromosomes in encysted dry eggs and nauplii. Proc. Japan Acad. 39, 306-309. RUSSO-CAIAS. & BELLINIL. (1960) Idrolisi enzimatica dell ATP nello sviluppo di ~lrtemia salina Leach diploide e tetraploide. 1st. Sci. Univ. Camerino, Rend. 1, 136-144. URBANI E. & BELLINI L. (1958) Studio delle ribonucleasi acida nella sviluppo di ~Irtemia salina Leach. Atti Accad. nazion. Lincei, Rend. 25, 198-201. WARNER A. H. & FINAMORE F. J. (1967) Nucleotide metabolism during brine shrimp embryogenesis, aT. biol. Chem. 242, 1933-1937. WARNERA. H. & MCCLEAYD. K. (1968) Studies on the biosynthesis and role of diguanosine tetraphosphate during the growth and development of Artemia salina. Devel. Biol. 18, 278-293. Key Word Index--Anoxia; anaerobiosis; lactic acid dehydrogenase; .xlrtemia salina; brine shrimp; development; carbohydrate metabolism during development; glycogen; trehalose; glycerol.