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Free amino acid changes in normal and eyestalkless megalopa larvae of the blue crab, Callinectes sapidus, during the course of the molt cycle
Comp. Biochem. Physiol., 1975, Vol. 51A, pp. 75 to 78. Pergamon Press. Printed in Great Britain
FREE AMINO ACID CHANGES IN NORMAL, AND EYESTALKLESS MEGALOPA LARVAE OF THE BLUE CRAB, CALLIiVECTES SAPIDUS, DURING THE COURSE OF THE MOLT CYCLE* ROBERT K. TucKERlt
AND JOHN D. COSTLOW, JR.~:
W.S. Department of Commerce, Middle Atlantic Coastal Fisheries Center, Sandy Hook Laboratory, Highlands, New Jersey 07732, U.S.A.; and la aDuke University Marine Laboratory, Beaufort, North Carolina 28516, U.S.A. (Received
21 January 1974)
Abstract-l. In normal megalopa larvae of Callinectes sapidus, the intracellular free amino compounds proline, taurine, alanine, glutamic acid and serine increased prior to the next molt. 2. In larvae with eyestalks removed on the day of molt to the megalopa stage, the increase in amino compounds, glutamic acid, proline, taurine and serine, occurred much sooner than in normals, paralleling the decrease in time to molt. 3. These changes are discussed in relation to molting, osmoregulation and possible hormonal involvement.
INTRODUCTION
normal and eyestalkless Cullinectes larvae during the course of the molting cycle, when the larvae were reared at a constant environmental salinity.
STUDIES of adult Crustacea, particularly the pioneering work of Florkin and his group, have implicated free amino acids as major components in isosmotic intracelhdar regulation when osmolarity of the hemolymph changes as a result of alterations in environmental salinity (Camien et al., 1951; Schoffeniels & G&s, 1970). Since the molting process in Crustacea involves water uptake, with attendant changes in osmotic balance, and is known to involve hormonal co-ordination (Carlisle & Knowles, 1959; Passano, 1961), it is of considerable interest to determine whether changes in free amino acids occur during the course of a molting cycle and whether eyestalk hormones may be involved in these changes. In larvae of the blue crab, Callinectes sapiaks, removal of both eyestalks within 12hr following molt to the megalopa stage considerably accelerates subsequent metamorphosis to the first crab stage (Costlow, 1963); eyestalk removal also increases hemolymph hyper-regulation in these larvae (Kalber & Costlow, 1968). The present study was undertaken to determine changes in concentrations of free amino acids and taurine in megalopa of
MATERIALS
AND METHODS
Larvae of C. sapidus were reared in the laboratory at a temperature of 25°C and salinity of 3Oy’ (parts per thousand) according to methods described by Costlow 8c Rookhout (1959). The removal of eyestalks in newly molted larvae was carried out on the day of molt to megalopa by the procedurepreviously described(Costlow, 1963). For amino acid analysis, pooled samples of three megalopa larvae were rinsed bristly with distilled water and blotted dry on a piece of filter paper. They were weighed to the nearest 0.1 mg, transferred to 1 ml water in a small test-tube and placed in a boiling water-bath for 10 mm to denature tissue proteolytic enzymes. After cooling, 15 w pentacldorophenol dissolved in ethyl alcohol (100 ~1) was added as a preservative and the tubes stored under refrigeration. At the time of analysis, 0.1 pmole nor-leucine (in 100 ~1 of buffer solution) was added as an internal standard and the larvae homogenized in a Potter-Elvehjem blender with approximately 2 ml 0.2 M citrate buffer, pH 2.2. The homogenate was transferred to a centrifuge tube, the volume adjusted to 3 ml with buffer, and particulate matter removed by centrifuaation for auuroximatelv 10 min at 2000 revlmin in a day-Adams -desk top centrifuge. Protein ‘was removed in an Amicon Ultrafiltration Unit, Model IO-PA, with a Type UM-10 membrane. Aliquots of the filtrate were analyzed on a Beckman Model 116 amino acid analyzer (Moore et al., 1958); concentrations of the free amino acids were calculated on the basis of pmoles/g wet weight of larvae.
* Portions of the work were supported by a contract between the office of Naval Research and Duke University, NR 104194. t Present address: U.S. Department of Commerce, National Marine Fisheries Service, NOAA, Sandy Hook Laboratory, Highlands, New Jersey. $ Present address: Duke University Marine Laboratory, North Carolina. 75
ROBERT K. TUCKER ANDJOHND. &mow,
76
RESULTS Concentrations of free amino acids and taurine in the pooled samples of larvae on specific days of the megalopa period are presented in Table 1. Concentrations are in pmoles/g wet weight of larvae. Day 0 represents the day on which the larvae molted to megalopa from the final zoeal stage. Day 0 was also the time when eyestalks were removed, after molt, from those animals so designated. The removal of eyestalks within 12 hr after the molt to the megalopa stage greatly accelerates the subsequent molt to the first crab stage. In an earlier study, under the same conditions of temperature and salinity, megalopa with eyestalks removed on day 0 took an average of 57 days to molt to the first crab stage as compared to 8.4 days for the normal controls (Costlow, 1963). Thus the time
JR.
periods, days 8-11 for normal and days 3-5 for eyestalkless larvae, are both late in the molt cycle for their respective groups. Data from these time periods have been averaged and are presented in Tabie 2, along with the averages for normal animals during the early part of the molt cycle, days O-4. In normal megalopa, late in the molt cycle, proline, taurine, alanine, glutamic acid and serine showed considerable elevations compared to concentrations early in the megalopa stage. The increases were greater than 70 per cent and, in the case of serine, the concentration had more than doubled. These changes are statistically significant (P ‘: 0.05, for alanine and proline, and P
Table 1. Concentrations of amino compounds, in pmoles/g wet weight, in megalopa larvae of C. supidus Normal
Eyestalkless
Late molt cycle
Late molt cycle
Normal
Early molt cycle Amino compound Alanine Aspartic acid Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
Day 0*
Day 1
Day 3
Day 4
Day8
Day9
Day11
Day 3
Day 4
Day 5
12.5 4.6 6.6 18.2 4.6 6.2 1.3 2.7 23.8 6.4 18.1 3.0 2.8 5.1
7.6 5.2 4.9 20.5 4.6 5.6 0.6 2.3 20.9 3.7 16.0 l-8
6.5 3.5 6.0 27.4 3.6 4.2 1.2 2.3 25.3 4.8 19.5 2.0 3.1 3.8
6.9 43
14.6 4.7 9.4 27.7 5.1 7.8
18.4 4.0 9.7 25.0 4.8 6.9
8.8 3.5
::.: 45.0 10.6 26.1 4.5 46 6.5
1.; 28.1 11.3 30.9 4.5 4.4 7.0
10.8 5.5 10.1 28.9 3.3 4.0 1.5 1.4 52.3 11.0 34.0 3.8 2.6 4.6
9.1 4.2 8.6 27.2 0.8 1.5 0.5 0.8 50.4 7.9 27.3 16 0.7 1.5
11.2 5.4 9.8 29.5 1.9 2.1 I.1 1.3 57.2 10.2 26,5 2.5 1.6 2.4
’ 2:.: 3.1 3.6 ;:; 27.5 4.8 151 ;:; -
2i.i 1.2 2.2 0.9
. 4:.;
. 2Z.9 2.1 1.7 2.2
* Day 0 is the day of molt from the final zoeal stage. Table 2. Concentration
of amino compounds in megalopa larvae of C. snpidus (pmoles/g wet weight of larvae, means+S.E.M.)
Amino compound
Normal Day 0*-4
Normal Day 8-l 1
Eyestalkless Day 3-5
Alanine Aspartic acid Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
8.42 1.4 4.4kO.4 5.6kO.4 23.5+24 4.OkO.4 49kO.6 0.9 * 0.2 2.3 +O.l 24.4 + 1.4 4.9 + 0.6 17.2+ 1.0 2.3+_03 3.0t0.1 4.4kO.4
14.6k2.2 4.750.4 9.7kO.l 27.2+ 1.2 4.4kO.6 6.2+ 1.1 1.4kO.l 2.5kO.6 41.8 + 7.2 11.0+0.2 30.5k2.1 4.3 f 0.2 3.9206 6.0f0.7
9.7kO.8 4.4kO.6 9.1 rto.4 27.3k1.2 1.3+_03 1.9+02 0.8 &-0.2 l.lkO.2 51.2k3.3 8.4+ 1.0 26.3kO.6 2.1 kO.3 1.3kO.3 2.OkO.3
* Day 0 is the day of molt from the final zoeal stage.
Free amino acid changes in the blue crab during the molt cycle the essential amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine and valine also show no significant differences in concentrations in late as compared to early megalopa. Threonine, an essential amino acid, appears to have increased during the molt cycle in normal animals (P< @OOS). In eyestalkless Cullinectes megalopa, glutamic acid, proline, taurine (P< O@OS), and serine (PC 0.05) increase strikingly during the molt cycle. The increase of these compounds occurs much earlier in the megalopa with eyestalks removed than in normal animals, thus paralleling the acceleration in molting. The slight increase in alanine concentration is not statistically significant; concentrations of aspartic acid, glycine, methionine and threonine show little change. The essential amino acids, isoleucine, leucine, phenylalanine, tyrosine and valine, were decreased somewhat in the eyestalkless animals compared to normal megalopa.
DISCUSSION In megalopa of C. supidus, the removal of eyestalks brings about an increase in the free amino compounds, proline, taurine, serine and glutamic acid, considerably sooner than occurs in the normal animals as they approach molt. Since the eyestalks contain endocrine organs and releasing sites (Carlisle & Knowles, 1959) it might be tempting to infer a hormonal effect on amino acid metabolism. Such an effect may not be direct, however, but may reflect osmotic changes in these animals. Studies with a number of species of adult crabs have established that hemolymph osmolarity increases prior to molt, thus contributing to the large water intake at ecdysis responsible for the increase in body size (Baumberger & Olmstead, 1928; Passano, 1961). Data from studies of larvae of Rhithropanopeus harrasii, Cardisoma guanhumi, as well as C. sapidus, confkm that the increase in hemolymph osmolarity prior to molt is characteristic of developmental stages also (Kalber & Costlow, 1966, 1968). In Crustacea with eyestalks removed, the increases in hemolymph osmolarity prior to molt are even greater than in normal animals, and greater amounts of water uptake and concomitantly larger size increases are noted (Scudamore, 1947). Greater size increases occur for larvae of Callinectes with eyestalks removed (Costlow, 1963). Changes in hemolymph osmolarity in Crustacea are mainly due to changes in concentration of inorganic ions; free amino acid levels are low in the hemolymph (Florkin, 1961). In Homurus, for example, free amino acid levels in hemolymph were less than 2 per cent of the levels found in muscle (Camien et al., 1951). Intracellularly, however, free amino acids reach quite high levels, contributing, in some cases, up to half of the measured osmotically active molecules (Schoffeniels & Gilles, 1970).
77
Florkin and his co-workers have for a number of years studied the role of free amino acids in Crustacea, as well as other invertebrate taxa, earlier using microbiological amino acid assay techniques (&mien et al., 1951) and later the more rapid chromatographic methods to establish the importance of free amino acids in intracellular isosmotic regulation (Schoffeniels & Gilles, 1970). With the advent of modern analytical techniques a number of other investigators have contributed to this field, and considerable information exists concerning amino acid changes in Crustacea subjected to variations in environmental salinity (Huggins & Munday, 1968). In the present study the crab larvae were not subjected to changes in external salinity; however, since hemolymph osmolarity increases prior to molt, intracellular free amino acid increases may have occurred by mechanisms similar to those operating when hemolymph changes are induced by environmental salinity changes. From studies with isolated nerves of Eriocheir sinensis, Schoffeniels (1960) has suggested that inorganic ions might affect enzymes involved in intracellular biosynthesis of amino acids. The slight increase in the essential amino acid, threonine, in normal megalopa, indicates that decreased catabolism might also be involved. Changes in rates of protein metabolism occurring toward the end of the molt cycle (Skinner, 1966) may also contribute to changes in amino acid concentrations, but since only certain amino acids increase, it seems unlikely that protein incorporation changes play a major role. Mechanisms of hormonal alteration of hemolymph osmolarity during the intermolt cycle remain to be explained, although evidence exists that changes in permeability, perhaps in the gut region, may be involved (Kamemoto et al., 1966; Mantel, 1968). Finally, although it is reasonable that amino acid changes may be secondary to changes in hemolymph inorganic ion concentration, more direct hormonal effects on amino acid metabolism should not be ruled out. REFERENCES BAUMBERGER J. P. & OHMSTEAD,J. M. (1928) Changes in the osmotic pressure and water content of crabs during the molt cycle. P/q&Z. Zoiil. 1, 531-544. CAMEN M. N., SARLETH., DUCHATEAUG. & FLORKIN M. (1951) Non-protein amino acids in muscle and blood of marine and fresh water Crustacea. J. biol. Chem. 193, 881-885. CARLISLE D. B. & KNOWLES K. (1959) Endocrine Confrol in Crustaceans. Cambridge University Press, London. GISTLOW J. D., JR. (1963) The effect of eyestalk extirpation on metamorphosis of megalops of the blue crab, Callinectes sapidus Rathbun. Gen. & compnr. Endocr. 3, 120-130. COSTLOW J. D., JR. & B~~KHOUT C. G. (1959) The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol, Bull. mar. biol. Lab., Woods Hole 116,373-396.
78
ROBERTK. TUCKERAND JOHN D. COSTLOW,JR.
FLORKIN M. (1961) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMANT. H.). Vol. 1. . pp. 141-159. AcademicPress, New York. .’ HUGGINS A. K. & MUNDAY K. A. (1968) Crustacean metabolism. In Advances in Comparative Physiology and Biochemistry (Edited by L0wENSTErNO.), Vol. 3, pp. 271-378. Academic Press, New York. KALBERF. A. & COSTLOWJ. D., JR. (1966) The ontogeny of osmoregulation and its neurosecretory control in the decapod crustacean, Bhithropanapeus harrisii (Gould). Am. Zoologist 6,221-229. KALBER F. A. & Cosr~ow J. D., JR. (1968) Osmoregulation in larvae of the land crab, Cardisoma guanhumi Latreille. Am. Zoologist 8, 41 l-416. KAMEMOTOF. I., KATO K. N. & TUCKER L. E. (1966) Neurosecretion and salt and water balance in the Annelida and Crustacea. Am. Zoologist 6, 213-219. MANTELL. H. (1968) The foregut of Gecarcinus lateralis as au organ of salt and water balance. Am. Zoologist 8, 433-442. Moons S., SPACKMAN D. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. Analyt. Chem. 30,1185-1190.
PASMNOL. M. (1961) Molting and its control. In The Physiology of Crustacea (Edited by WATERMANT. H.), Vol. 1, pp. 473-536. Academic Press, New York. SCHOFFENIELS E. (1960) Origine des acides amines intervenant dans la regulation de la pression osmotique intracellulaire de Eriocheir sinensis Milne-Edwards. Archs int. Physiol. Biochem. 68,696698. SCHOPFENIELS E. & GILLES R. (1970) Osmoregulation in aquatic arthropods. In Chemical’Zoology -@dited by FLORKIN M. & SCHEERB. T.), Vol. 5, pp. 255-286. Academic Press, New York. SCUDAMOR~H. H. (1947) The infhrence of the sinus glands upon molting and associated changes in the crayfish. -Physiol. Zo&. 20, 187-208. SKINNERD. M. (1966) Breakdown and reformation of somatic muscle‘durihg the molt cycle of the land crab, Gecarcinus lateralis. J. exp. Zool. 163, 115-123. SNEDECORG. W. & CXHRAN W. G. (1967) Statistical Methods, 6th Edu. Iowa State University Press, Ames, Iowa. Key Word Index-Crab ; Callinectes sapidus; crustacean larvae; megalopa; osmoregulation; free amino acids; taurine; molt cycle; eyestalk removal.
FREE AMINO ACID CHANGES IN NORMAL, AND EYESTALKLESS MEGALOPA LARVAE OF THE BLUE CRAB, CALLIiVECTES SAPIDUS, DURING THE COURSE OF THE MOLT CYCLE* ROBERT K. TucKERlt
AND JOHN D. COSTLOW, JR.~:
W.S. Department of Commerce, Middle Atlantic Coastal Fisheries Center, Sandy Hook Laboratory, Highlands, New Jersey 07732, U.S.A.; and la aDuke University Marine Laboratory, Beaufort, North Carolina 28516, U.S.A. (Received
21 January 1974)
Abstract-l. In normal megalopa larvae of Callinectes sapidus, the intracellular free amino compounds proline, taurine, alanine, glutamic acid and serine increased prior to the next molt. 2. In larvae with eyestalks removed on the day of molt to the megalopa stage, the increase in amino compounds, glutamic acid, proline, taurine and serine, occurred much sooner than in normals, paralleling the decrease in time to molt. 3. These changes are discussed in relation to molting, osmoregulation and possible hormonal involvement.
INTRODUCTION
normal and eyestalkless Cullinectes larvae during the course of the molting cycle, when the larvae were reared at a constant environmental salinity.
STUDIES of adult Crustacea, particularly the pioneering work of Florkin and his group, have implicated free amino acids as major components in isosmotic intracelhdar regulation when osmolarity of the hemolymph changes as a result of alterations in environmental salinity (Camien et al., 1951; Schoffeniels & G&s, 1970). Since the molting process in Crustacea involves water uptake, with attendant changes in osmotic balance, and is known to involve hormonal co-ordination (Carlisle & Knowles, 1959; Passano, 1961), it is of considerable interest to determine whether changes in free amino acids occur during the course of a molting cycle and whether eyestalk hormones may be involved in these changes. In larvae of the blue crab, Callinectes sapiaks, removal of both eyestalks within 12hr following molt to the megalopa stage considerably accelerates subsequent metamorphosis to the first crab stage (Costlow, 1963); eyestalk removal also increases hemolymph hyper-regulation in these larvae (Kalber & Costlow, 1968). The present study was undertaken to determine changes in concentrations of free amino acids and taurine in megalopa of
MATERIALS
AND METHODS
Larvae of C. sapidus were reared in the laboratory at a temperature of 25°C and salinity of 3Oy’ (parts per thousand) according to methods described by Costlow 8c Rookhout (1959). The removal of eyestalks in newly molted larvae was carried out on the day of molt to megalopa by the procedurepreviously described(Costlow, 1963). For amino acid analysis, pooled samples of three megalopa larvae were rinsed bristly with distilled water and blotted dry on a piece of filter paper. They were weighed to the nearest 0.1 mg, transferred to 1 ml water in a small test-tube and placed in a boiling water-bath for 10 mm to denature tissue proteolytic enzymes. After cooling, 15 w pentacldorophenol dissolved in ethyl alcohol (100 ~1) was added as a preservative and the tubes stored under refrigeration. At the time of analysis, 0.1 pmole nor-leucine (in 100 ~1 of buffer solution) was added as an internal standard and the larvae homogenized in a Potter-Elvehjem blender with approximately 2 ml 0.2 M citrate buffer, pH 2.2. The homogenate was transferred to a centrifuge tube, the volume adjusted to 3 ml with buffer, and particulate matter removed by centrifuaation for auuroximatelv 10 min at 2000 revlmin in a day-Adams -desk top centrifuge. Protein ‘was removed in an Amicon Ultrafiltration Unit, Model IO-PA, with a Type UM-10 membrane. Aliquots of the filtrate were analyzed on a Beckman Model 116 amino acid analyzer (Moore et al., 1958); concentrations of the free amino acids were calculated on the basis of pmoles/g wet weight of larvae.
* Portions of the work were supported by a contract between the office of Naval Research and Duke University, NR 104194. t Present address: U.S. Department of Commerce, National Marine Fisheries Service, NOAA, Sandy Hook Laboratory, Highlands, New Jersey. $ Present address: Duke University Marine Laboratory, North Carolina. 75
ROBERT K. TUCKER ANDJOHND. &mow,
76
RESULTS Concentrations of free amino acids and taurine in the pooled samples of larvae on specific days of the megalopa period are presented in Table 1. Concentrations are in pmoles/g wet weight of larvae. Day 0 represents the day on which the larvae molted to megalopa from the final zoeal stage. Day 0 was also the time when eyestalks were removed, after molt, from those animals so designated. The removal of eyestalks within 12 hr after the molt to the megalopa stage greatly accelerates the subsequent molt to the first crab stage. In an earlier study, under the same conditions of temperature and salinity, megalopa with eyestalks removed on day 0 took an average of 57 days to molt to the first crab stage as compared to 8.4 days for the normal controls (Costlow, 1963). Thus the time
JR.
periods, days 8-11 for normal and days 3-5 for eyestalkless larvae, are both late in the molt cycle for their respective groups. Data from these time periods have been averaged and are presented in Tabie 2, along with the averages for normal animals during the early part of the molt cycle, days O-4. In normal megalopa, late in the molt cycle, proline, taurine, alanine, glutamic acid and serine showed considerable elevations compared to concentrations early in the megalopa stage. The increases were greater than 70 per cent and, in the case of serine, the concentration had more than doubled. These changes are statistically significant (P ‘: 0.05, for alanine and proline, and P
Table 1. Concentrations of amino compounds, in pmoles/g wet weight, in megalopa larvae of C. supidus Normal
Eyestalkless
Late molt cycle
Late molt cycle
Normal
Early molt cycle Amino compound Alanine Aspartic acid Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
Day 0*
Day 1
Day 3
Day 4
Day8
Day9
Day11
Day 3
Day 4
Day 5
12.5 4.6 6.6 18.2 4.6 6.2 1.3 2.7 23.8 6.4 18.1 3.0 2.8 5.1
7.6 5.2 4.9 20.5 4.6 5.6 0.6 2.3 20.9 3.7 16.0 l-8
6.5 3.5 6.0 27.4 3.6 4.2 1.2 2.3 25.3 4.8 19.5 2.0 3.1 3.8
6.9 43
14.6 4.7 9.4 27.7 5.1 7.8
18.4 4.0 9.7 25.0 4.8 6.9
8.8 3.5
::.: 45.0 10.6 26.1 4.5 46 6.5
1.; 28.1 11.3 30.9 4.5 4.4 7.0
10.8 5.5 10.1 28.9 3.3 4.0 1.5 1.4 52.3 11.0 34.0 3.8 2.6 4.6
9.1 4.2 8.6 27.2 0.8 1.5 0.5 0.8 50.4 7.9 27.3 16 0.7 1.5
11.2 5.4 9.8 29.5 1.9 2.1 I.1 1.3 57.2 10.2 26,5 2.5 1.6 2.4
’ 2:.: 3.1 3.6 ;:; 27.5 4.8 151 ;:; -
2i.i 1.2 2.2 0.9
. 4:.;
. 2Z.9 2.1 1.7 2.2
* Day 0 is the day of molt from the final zoeal stage. Table 2. Concentration
of amino compounds in megalopa larvae of C. snpidus (pmoles/g wet weight of larvae, means+S.E.M.)
Amino compound
Normal Day 0*-4
Normal Day 8-l 1
Eyestalkless Day 3-5
Alanine Aspartic acid Glutamic acid Glycine Isoleucine Leucine Methionine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine
8.42 1.4 4.4kO.4 5.6kO.4 23.5+24 4.OkO.4 49kO.6 0.9 * 0.2 2.3 +O.l 24.4 + 1.4 4.9 + 0.6 17.2+ 1.0 2.3+_03 3.0t0.1 4.4kO.4
14.6k2.2 4.750.4 9.7kO.l 27.2+ 1.2 4.4kO.6 6.2+ 1.1 1.4kO.l 2.5kO.6 41.8 + 7.2 11.0+0.2 30.5k2.1 4.3 f 0.2 3.9206 6.0f0.7
9.7kO.8 4.4kO.6 9.1 rto.4 27.3k1.2 1.3+_03 1.9+02 0.8 &-0.2 l.lkO.2 51.2k3.3 8.4+ 1.0 26.3kO.6 2.1 kO.3 1.3kO.3 2.OkO.3
* Day 0 is the day of molt from the final zoeal stage.
Free amino acid changes in the blue crab during the molt cycle the essential amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine and valine also show no significant differences in concentrations in late as compared to early megalopa. Threonine, an essential amino acid, appears to have increased during the molt cycle in normal animals (P< @OOS). In eyestalkless Cullinectes megalopa, glutamic acid, proline, taurine (P< O@OS), and serine (PC 0.05) increase strikingly during the molt cycle. The increase of these compounds occurs much earlier in the megalopa with eyestalks removed than in normal animals, thus paralleling the acceleration in molting. The slight increase in alanine concentration is not statistically significant; concentrations of aspartic acid, glycine, methionine and threonine show little change. The essential amino acids, isoleucine, leucine, phenylalanine, tyrosine and valine, were decreased somewhat in the eyestalkless animals compared to normal megalopa.
DISCUSSION In megalopa of C. supidus, the removal of eyestalks brings about an increase in the free amino compounds, proline, taurine, serine and glutamic acid, considerably sooner than occurs in the normal animals as they approach molt. Since the eyestalks contain endocrine organs and releasing sites (Carlisle & Knowles, 1959) it might be tempting to infer a hormonal effect on amino acid metabolism. Such an effect may not be direct, however, but may reflect osmotic changes in these animals. Studies with a number of species of adult crabs have established that hemolymph osmolarity increases prior to molt, thus contributing to the large water intake at ecdysis responsible for the increase in body size (Baumberger & Olmstead, 1928; Passano, 1961). Data from studies of larvae of Rhithropanopeus harrasii, Cardisoma guanhumi, as well as C. sapidus, confkm that the increase in hemolymph osmolarity prior to molt is characteristic of developmental stages also (Kalber & Costlow, 1966, 1968). In Crustacea with eyestalks removed, the increases in hemolymph osmolarity prior to molt are even greater than in normal animals, and greater amounts of water uptake and concomitantly larger size increases are noted (Scudamore, 1947). Greater size increases occur for larvae of Callinectes with eyestalks removed (Costlow, 1963). Changes in hemolymph osmolarity in Crustacea are mainly due to changes in concentration of inorganic ions; free amino acid levels are low in the hemolymph (Florkin, 1961). In Homurus, for example, free amino acid levels in hemolymph were less than 2 per cent of the levels found in muscle (Camien et al., 1951). Intracellularly, however, free amino acids reach quite high levels, contributing, in some cases, up to half of the measured osmotically active molecules (Schoffeniels & Gilles, 1970).
77
Florkin and his co-workers have for a number of years studied the role of free amino acids in Crustacea, as well as other invertebrate taxa, earlier using microbiological amino acid assay techniques (&mien et al., 1951) and later the more rapid chromatographic methods to establish the importance of free amino acids in intracellular isosmotic regulation (Schoffeniels & Gilles, 1970). With the advent of modern analytical techniques a number of other investigators have contributed to this field, and considerable information exists concerning amino acid changes in Crustacea subjected to variations in environmental salinity (Huggins & Munday, 1968). In the present study the crab larvae were not subjected to changes in external salinity; however, since hemolymph osmolarity increases prior to molt, intracellular free amino acid increases may have occurred by mechanisms similar to those operating when hemolymph changes are induced by environmental salinity changes. From studies with isolated nerves of Eriocheir sinensis, Schoffeniels (1960) has suggested that inorganic ions might affect enzymes involved in intracellular biosynthesis of amino acids. The slight increase in the essential amino acid, threonine, in normal megalopa, indicates that decreased catabolism might also be involved. Changes in rates of protein metabolism occurring toward the end of the molt cycle (Skinner, 1966) may also contribute to changes in amino acid concentrations, but since only certain amino acids increase, it seems unlikely that protein incorporation changes play a major role. Mechanisms of hormonal alteration of hemolymph osmolarity during the intermolt cycle remain to be explained, although evidence exists that changes in permeability, perhaps in the gut region, may be involved (Kamemoto et al., 1966; Mantel, 1968). Finally, although it is reasonable that amino acid changes may be secondary to changes in hemolymph inorganic ion concentration, more direct hormonal effects on amino acid metabolism should not be ruled out. REFERENCES BAUMBERGER J. P. & OHMSTEAD,J. M. (1928) Changes in the osmotic pressure and water content of crabs during the molt cycle. P/q&Z. Zoiil. 1, 531-544. CAMEN M. N., SARLETH., DUCHATEAUG. & FLORKIN M. (1951) Non-protein amino acids in muscle and blood of marine and fresh water Crustacea. J. biol. Chem. 193, 881-885. CARLISLE D. B. & KNOWLES K. (1959) Endocrine Confrol in Crustaceans. Cambridge University Press, London. GISTLOW J. D., JR. (1963) The effect of eyestalk extirpation on metamorphosis of megalops of the blue crab, Callinectes sapidus Rathbun. Gen. & compnr. Endocr. 3, 120-130. COSTLOW J. D., JR. & B~~KHOUT C. G. (1959) The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol, Bull. mar. biol. Lab., Woods Hole 116,373-396.
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ROBERTK. TUCKERAND JOHN D. COSTLOW,JR.
FLORKIN M. (1961) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMANT. H.). Vol. 1. . pp. 141-159. AcademicPress, New York. .’ HUGGINS A. K. & MUNDAY K. A. (1968) Crustacean metabolism. In Advances in Comparative Physiology and Biochemistry (Edited by L0wENSTErNO.), Vol. 3, pp. 271-378. Academic Press, New York. KALBERF. A. & COSTLOWJ. D., JR. (1966) The ontogeny of osmoregulation and its neurosecretory control in the decapod crustacean, Bhithropanapeus harrisii (Gould). Am. Zoologist 6,221-229. KALBER F. A. & Cosr~ow J. D., JR. (1968) Osmoregulation in larvae of the land crab, Cardisoma guanhumi Latreille. Am. Zoologist 8, 41 l-416. KAMEMOTOF. I., KATO K. N. & TUCKER L. E. (1966) Neurosecretion and salt and water balance in the Annelida and Crustacea. Am. Zoologist 6, 213-219. MANTELL. H. (1968) The foregut of Gecarcinus lateralis as au organ of salt and water balance. Am. Zoologist 8, 433-442. Moons S., SPACKMAN D. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. Analyt. Chem. 30,1185-1190.
PASMNOL. M. (1961) Molting and its control. In The Physiology of Crustacea (Edited by WATERMANT. H.), Vol. 1, pp. 473-536. Academic Press, New York. SCHOFFENIELS E. (1960) Origine des acides amines intervenant dans la regulation de la pression osmotique intracellulaire de Eriocheir sinensis Milne-Edwards. Archs int. Physiol. Biochem. 68,696698. SCHOPFENIELS E. & GILLES R. (1970) Osmoregulation in aquatic arthropods. In Chemical’Zoology -@dited by FLORKIN M. & SCHEERB. T.), Vol. 5, pp. 255-286. Academic Press, New York. SCUDAMOR~H. H. (1947) The infhrence of the sinus glands upon molting and associated changes in the crayfish. -Physiol. Zo&. 20, 187-208. SKINNERD. M. (1966) Breakdown and reformation of somatic muscle‘durihg the molt cycle of the land crab, Gecarcinus lateralis. J. exp. Zool. 163, 115-123. SNEDECORG. W. & CXHRAN W. G. (1967) Statistical Methods, 6th Edu. Iowa State University Press, Ames, Iowa. Key Word Index-Crab ; Callinectes sapidus; crustacean larvae; megalopa; osmoregulation; free amino acids; taurine; molt cycle; eyestalk removal.