Neuroendocrine control of nitrogen metabolism in the indian field crab Oziotelphusa s. senex fabricius—II. Enzyme activities

Comp. Biochem. Physiol. Vol. 71B, pp. 223 to 228, 1982 Printed in Great Britain. All rights reserved

0305-0491/82/020223-06503.00/0 Copyright © 1982 Pergamon Press Ltd

N E U R O E N D O C R I N E CONTROL OF NITROGEN METABOLISM IN THE INDIAN FIELD CRAB O Z I O T E L P H U S A S. SENEX FABRICIUS--II. ENZYME ACTIVITIES R. RAMAMURTHI*, K . RAGHAVAIAHt, V. CHANDRA SEKHARAM a n d

B. T. SCHEER~ Department of Zoology, Sri Venkateswara University, Tirupati 517 502 (A.P.) India (Received 18 M a y 1981)

Abstract 1. The increase in elimination of NPN and of concentration of NPN in tissues, primarily in the form of NH3, reported earlier (Comp. Biochem. Physiol. 67B, 437, 1980) as a consequence of extirpation of eyestalks from this fresh-water crab, is here traced to inhibition, by neurohumors from the eyestalks, of enzymes producing NH 3 by hydrolysis of amides and oxidation of amino acids. 2. Glutaminase activity was inhibited by an eyestalk factor, in haemolymph (HL), hepatopancreas (HP), muscle (M), hypodermis (HD), and gill (G). Asparaginase activity was inhibited only in HP, HD, and G. Activity of glutamate dehydrogenase was inhibited in M, HD and G. 3. The activities of ala and asp aminotransferases were stimulated in HP, M, HD and G. 4. No general pattern of action on oxidoreductases in general was demonstrated, or on activity of arginase, xanthine dehydrogenase, protease, or ribonuclease, although effects on some of these were evident in some tissues. 5. We conclude that the neuroendocrine system of the eyestalk of this crab acts during intermoult to conserve nitrogen stores and to promote turnover of amino acids, through direct action on the enzymes involved.

INTRODUCTION

In the first paper of this series (Raghavaiah et al., 1980), we presented results showing that extirpation of eyestalks, and injection of eyestalk extracts into eyestalkless crabs, altered elimination and tissue concentrations of nitrogenous c o m p o u n d s in a pattern which suggested that, during the intermoult stage of the moulting cycle of animals of this species, eyestalk factor(s) act to conserve nitrogen by limiting the catabolism of amino acids and purines. The present paper reports results of tests of our former conclusions by means of enzyme assays and studies of the effects of extirpation and extracts of eyestalks on the activities of enzymes of nitrogen metabolism. MATERIALS AND METHODS

The Indian fresh-water field crab Oziotelphusa s. senex Fabricius was used in the same experimental design used earlier. After acclimation of animals to laboratory conditions, eyestalks were extirpated from 3/4 of the animals in any experimental group, reserving the others as unoperated controls. Eyestalkless animals were removed from an experimental group for dissection of tissues and enzyme assay at intervals of 1, 2, 3, 5 and 7 days post-operative. At 7 days, animals were injected, 12 hr before dissection, either with crustacean saline (Van Harreveld, 1936) or with an * Requests for reprints should be addressed to Prof. Ramamurthi at Tirupati. "t"Based on a thesis submitted by K. Raghavaiah to S. V. University for the Ph.D. degree. :~Westmont College, Santa Barbara, California; mailing address 1905 Mission Ridge Rd., Santa Barbara, CA 93103, U.S.A. 223

alcoholic extract of eyestalks in saline, prepared according to Silverthorn (1976). On removal of any animal for dissection, a sample of haemolymph was aspirated and other tissues hepatopancreas, muscle, hypodermis, and gill were quickly dissected out, weighed, and prepared for assay. The following enzymes (nomenclature, IUB, 1978) were assayed by methods of the authors cited. Aspartate aminotransferase, L-aspartate: 2-oxoglutarate aminotransferase, 2.6.1.1. Alanine aminotransferase, DL-alanine: 2-oxoglutarate aminotransferase, 2.6.1.2 Reitman & Frankel (1957). Glutamate dehydrogenase (NAD+), 1.4.1.3 Prameelamma & Swami (1975). L-glutaminase, t,-glutamine amidohydrolase, 3.5.1.2. L-asparaginase, L-asparagine amidohydrolase, 3.5.1.1 Meister (1955). Glutamine synthetase, L-glutamate: ammonia ligase (ADP), 6.3.1.2 Wu (1963), modified. Arginase, L-arginine amidinohydrolase, 3.5.3.1 Campbell (1961), mod. according to Brown & Cohen (1959) and Natelson (1971). Urease, urea amidohydrolase, 3.5.1.5 Hoffmann & Teicher, (according to Bergmayer (1965). Protease, Moore & Stein (1968). Ribonuclease, RNase, 3.1.27.5 McDonald (1955), Tuve & Anfinsen (1960) according to Bagi and Farkas (1967). Adenosinetriphosphatase. ATPase, ATP phosphohydrolase, 3.6.1.3 Potter (1939). Cytochrome Oxidase, Oda et al. (1958). Malate dehydrogenase, L-malate: NAD + oxidoreductase, 1.1.1.37 Succinate dehydrogenase, succinate oxidoreductase, 1.3.99.1 Nachlas et al. (1960), modified by Prameelamma et al. (1975) For determination of adenine nucleotides, tissues were excised, blotted, and dropped into liquid N2 within 2 min. The frozen tissues were homogenized with 3 vol of ice-cold 7~o trichloroacetic acid. The precipitate was separated in

R. RAMAMURTHIet al.

224

Table 1. Activity, mg product-(mg protein, hr)- ~, of enzymes of storage and release of NH3 in tissues of O. s e n e x

Time (days), post-operative Tissue

Enzyme Glutaminase

Haemolymph Hepatopancreas Muscle Hypodermis Gill

Asparaginase

Hepatopancreas Muscle Hypodermis Gill

0 (unop.)

1

2

3

5

7 saline

7 ESE

200.7 ± 39.2 688.1 ± 138.1 433.1 ± 75.4 587.2 ± 44.8 835 ± 94

358.5* ± 43.3 966.7* ± 24 559.3* ± 44.1 693.4* ± 17.2 1172" ± 41

314.3* ± 25.6 906.6* _+ 48.7 497.7 ± 18.1 667.6* ± 23.5 1100" ± 19

320.6* ± 17.4 867.5* _+ 32.8 478.4 ± 14 671.3" ± 37.8 993.4* ± 58.7

367.2* _+ 26.1 977.5* ± 47.6 523.8* ± 12.6 723.5* ± 13 1030" ± 37

398.2* _+28 1158" ± 78 523.5* _+ 14.4 801' ± 15.3 1178' + 25

213.7t _+ 19.7 703.91 _+ 61 466.9+ _+ 13.9 625.5+ ± 50.3 1018" + 109

173.4 ± 7.5 75.3 ± 8.6 117.4 ± 5.2 234.9 ± 12.6

195.5 ± 17.3 74.7 ± 7.8 138.2" ± 8.4 249.9 ± 20.1

196.3" ± 14.2 77.8 ± 5.5 124.5 ± 5 147.9 ± 17.3

188.l* ± 6.9 79.3 ± 5.1 122.8 ± 4.8 256.8 ±13.7

228.7* ± 9.7 79.9 ± 7.l 133.6" ± 8.2 283.6* ± 14.2

225.2* ± 6.7 91.8 + 10.3 153.5" ± 8.8 285.1 * ± 13.8

190.5t ± 12.8 79.5 ± 4.4 128.8-t + 10.1 249¢ + 8

Glutamine synthetase

Hepatopancreas

70.1 ± 10.7

80.6 ± 11.9

76.7 ± 17

79. l ± 17

89.8 ± 16.4

95.1 + 19.7

76.6 + 14.9

Glutamate dehydrogenase

Haemolymph

200.7 +_ 39.2 64.2 + 16.1 225.5 _+ 41.4 56.7 ± 11.6 137.2 ± 13.9

358.5* + 43.3 49 ± 12 306 _+ 41.9 99.1" ± 16.9 211.8" ± 28.5

314.3" + 25.6 56.7 ± 11.3 298.2* ± 22.1 102.3' ± 16.7 216.9" ± 22.8

320.6* +17.4 48.2 ± 10.1 292* _+ 21.7 105.7" ± 23 201' ± 20

367.2* _+ 26.1 45.1 ± 16.4 321.8" ± 40.7 101.3" ± 11.8 199.4" ± 20.7

398.6* + 28 46.2 +_ 13.1 324.6* + 38.8 118.1" ± 22.4 238.8* ± 25

213.7t _+ 19.7 50.1 +_ I1.1 238.7t ± 6 55.9+ _+ 6.6 147.1+ ± 13.3

Hepatopancreas Muscle Hypodermis Gill

As influenced by extirpation of, and injection of extracts (ESE) of, eyestalks. Means for groups of 8 animals + 1~ confidence limits.

the centrifuge, and the supernatant fluid was neutralized with 5 M K2CO 3. The nucleotides were ddtermined in the neutral supernatant by the method of Cohn (1957). RESULTS We earlier reported (Raghavaiah e t al., 1980) an immediate and persistent increase in elimination of total non-protein nitrogen (NPN) after extirpation of eyestalks, which was immediately reversed by injection of eyestalk extract (ESE). The principal comp o n e n t of the N P N was NH3, but elimination of the other classical end-products of nitrogen metabolism, urea a n d uric acid, in relatively small quantities, followed the pattern of change of total N P N . The criteria for neuroendocrine control used earlier, namely a change from unoperated control values after extirpation of eyestalks, with reversal of the change immediately after injection of ESE, will be used here as the criterion of neuroendocrine influence on enzyme activity. The criterion of significant change will be, as before, the method of statistical confidence limits. Changes which are not reversed by ESE may be interpreted as resulting from operative trauma.

The changes in activity of the enzymes which release N H 3 by hydrolysis of amides or oxidation of a m i n o acids (Table 1) are consistent with the earlier results. The activity of glutaminase is inhibited during the intermoult stage of the moulting cycle of O. s e n e x by eyestalk factors. Inhibition of asparaginase is less marked, but occurs in hepatopancreas, hypodermis and gill. G l u t a m a t e dehydrogenase is also inhibited, in all tissues except hepatopancreas. G l u t a m i n e synthetase, which synthesizes glutamine from glutamate and N H 3, was detected only in hepatopancreas, where its activity was not influenced by extirpation or extracts of eyestalks. Urea N is a m i n o r c o m p o n e n t of total N P N in this crab, but the elimination of urea increased after extirpation and was restored by ESE. We proposed that the source of this urea was hydrolysis of arginine, since there is no evidence that urea synthesis from N H 3 occurs in crustaceans. The results in Table 2 show that arginase is inhibited during intermoult by an eyestalk factor in hepatopancreas and hypodermis. This could account for the earlier results. Uric acid N is an even smaller c o m p o n e n t of total N P N than is urea N, and the evidence for neuroendocrine control

Nitrogen metabolism in the Indian field crab

225

Table 2. Specific activities, mg product.mg-1, hr-~, in tissues of O. senex, of enzymes producing urea (arginase) and uric acid (xanthine dehydrogenase), as for Table 1 Time (days), post-operative Enzyme Arginase

Tissue Hepatopancreas Muscle Hypodermis Gill

Xanthine dehydrogenase

Hepatopancreas Muscle Hypodermis Gill

0 (unop.)

1

2

3

5

7 saline

7 ESE

1100 ± 33 469.6 ± 42.9 2186 _+ 47 2500 _+75

1186" _+ 39 472.2 _+ 66.7 2276 _+ 49 2522 _+44

1123 + 44 458.6 _+ 40.6 2165 _+ 36 2492 _+79

1148 _+ 57 475.4 _+ 19.9 2220 _+ 43 2492 +_95

1319" _+ 72 482.4 _+ 37.4 2306 _+ 100 2521 +86

1334" _+ 71 500.2 _+ 32.8 2341" _+ 56 2505 _+74

1166t _+35 467.9 _+ 15.6 2207t +_ 32 2497 _+33

521.8 _+ 31.1 327.6 + 41.2 702.6 _+ 95.3 174.6 _+ 28.6

601.8" _+ 18 339.9 _+ 41.7 1193" _+ 41.7 168.2 _+ 40.7

584.4 +_ 36.9 318.2 _+ 35.8 902.4* _+ 91.3 174.7 _+ 22.9

573.6 + 37.2 330.9 _+ 58.4 922* +_ 43.6 188.6 +_ 58.5

549.5 _+ 32.8 338.1 _+ 49 942* ± 64.4 167.6 +_ 28.4

595.6* _+ 22.1 360.6 _+ 47.2 1149" _+ 208 186.6 _+ 55.9

558.8t _+ 9.3 337.6 _+ 15.9 942.8* ± 35.6 172.3 _+ 113

* > Unoperated controls. t < Saline controls. of its formation and elimination is equivocal. The activity of xanthine dehydrogenase (Table 2) in hepatopancreas increased on day 1 and 7 post-operative, and the level was restored to within the confidence interval of unoperated controls by ESE. The marked postoperative increase in activity in hypodermis was not reversed by ESE, and no significant changes occurred in other tissues. We earlier proposed that the increase in elimination of N P N after eyestalk extirpation may have resulted from increased catabolism of protein and nucleic acids, and some decreases in concentration of protein in muscle, hypodermis, and gill were evident post-operatively. However, the results in Table 3 show that the only significant change in protease activity after extirpation was an increase in hepatopancreas. Injection of ESE resulted in a decrease which was not sufficient to return protease activity to the confidence range for unoperated controls. Activities of ribonuclease were not changed in any tissue. The effects of extirpation on the activity of the aminotransferases were unique. The activities of the two enzymes assayed decreased post-operatively in all the solid tissues, and were returned to levels of unoperated controls, and above those of saline-injected controls by injection of ESE. This demonstrates a stimulation of these enzymes by an eyestalk factor during intermoult. To determine whether the effects of eyestalk extirpation are related to effects on general metabolism, the concentrations of the adenine nucleotides in the tissues were studied (Table 4). The only significant change attributable to an eyestalk factor was a small and delayed increase in concentration of A D P in hepatopancreas and muscle, restored to unoperated control levels by ESE. The increase in gills, although immediate, was not reversed completely by ESE. The only comparable effect on concentration of ATP was a decrease in hepatopancreas on day 5 and 7 which

was not reversed by ESE. Similarly, the delayed increases in activity of ATPase in hepatopancreas and muscle were not sufficiently influenced by ESE to support an inference of any control over this enzyme or the concentration of its substrate and product. The only clear-cut evidence for eyestalk control of oxidative enzymes in general (Table 5) was a marked post-operative increase in the activity of succinate dehydrogenase in hepatopancreas, returned to control levels by ESE. Similar increases in muscle and hypodermis were not influenced by ESE. The change in activity of succinate dehydrogenase is evidently not correlated with a general change in activity of the Krebs TCA cycle, since malate dehydrogenase activity was not affected. Likewise, terminal oxidation through the electron transport system is not affected, at least as regards cytochrome oxidase, the activity of which only showed a post-operative increase, which was not reversed by ESE, in muscle. CONCLUSIONS The results presented here show an effect of eyestalk factors, presumably neuroendocrine in nature, on the activity of certain enzymes of nitrogen metabolism during the intermoult stage of the intermoult cycle of O. s e n e x . Briefly, these effects amount to decreases in the production of NH3 by oxidation (Glutamate dehydrogenase activity) and by the hydrolysis of amides (glutaminase and asparaginase activities), and to increases in the turnover of nitrogen in the amino groups of amino acids (activity of aminotransferases). This is consistent with the general conclusion expressed earlier (Raghavaiah e t al., 1980) that the neuroendocrine system of the eyestalk acts to prevent loss of N during intermoult. All the solid tissues studied participate in this activity in some degree, and it is effected through direct control over the activity of the enzymes noted, rather than indirectly through an

226

R. RAMAMURTHIet al.

Table 3. Specific activities, in tissues of O. s e n e x , of protease (nM t y r o s i n e . m g - ~ .hr 1), amino transferases (nM p y r u v a t e . m g - 1 , hr-1) and ribonuclease (RNase), as for previous tables Time (hr), post-operative

Enzyme Protease

Tissues Hepatopancreas Muscle Hypodermis Gill

Aspartate amino transferase

Haemolymph Hepatopancreas Muscle Gill

Haemolymph

Alanine amino transferase

Hepatopancreas Muscle Hypodermis Gill

RNase

Hepatopancreas Muscle Hypodermis Gill

* > t < +< §>

Unoperated controls. Saline controls. Unoperated controls. Saline controls.

0 (unop.) 0.905 ± 0.062 0.59 ±_ 0.12 0.21 ± 0.07 0.43 ± 0.10

1

1.54' + 0,35 0,99* _+ 0.14 0,32 _+ 0~06 0.42 --

2

1.32" ± 0.33 0.59 ± 0.11 0.21 ± 0.07 0.46

3

5

1.74" ± 0.17 0.65 ± 0.09 0.31 ± 0.15 0.48 --

1.73" ± 0.36 0.56 ± 0.06 0.28 ± 0.03 0.45

7 saline 1.96" _+ 0.56 0.63 ± 0.07 0.32* ± 0.03 0.45 ± O.lO

7 ESE l.l 3+ ± 0.14 0.60 +_ 0.04 0.29 +_ 0.03 0.43 + 0.04

7.27 ± 1.07 143.6 ± 15.9 198.4 ± 8.3 101.6 + 7.6

8.36 + 1.51 161.4 ± 9.8 198.7 ± 10.1 83.3++ ± 9.1

6.6 ± 1.0 124.9 ± 14.5 181.9 ± 10.1 78.4~: _+ 6.2

5.35 ± 1.01 113.72~ ± 6.6 181~ ± 8.5 60.4++ _+ 10.3

6.3 ± 0.8 105.8:~ ± 11.8 158.8++ ± 14 58.8++ _+ 7.2

6.53 ± 1.35 100.9++ +_ 10.5 156.9:~ ± 12.4 66.2++ ± 5.2

5.91 ±1 140.3§ + 9.3 183.2§ ± 11.8 85.2§ ± 9.9

11.2 ± 1.8 243.8 ± 19.2 292 ± 13.8 161.9 ± 17.2 169.5 ± 6.5

13.1 _+ 2.4 254.6 ± 16.7 290.9 ± 15.9 155 +_ 13.5 158.2 +_ 12.3

10.3 + 1.5 204.3~ +_ 11.2 271.6 ± 9.8 143 +_ 13.6 152.1:[: ± 9.9

10.4 ± 1.5 192.7++ _+ 12.3 250.4++ +_ 7.4 143.4 ± 12.4 146.9++ ± 11.9

10.2 +_ 1.2 193.9++ _+ 14.1 225.7++ ± 11.4 131.8~: +_ 9.3 128.9++ ± 11.7

10.6 ± 2 190.6++ _+ 14.7 225.4++ ± 6.6 112.7++ + 10.1 125.2++ ± 5.4

9.93 + 1.21 239.2§ ± 24.1

9.2 +_ 2.4 4.4 +_ 1.4 4.8 ± 1.6 7 _+ 2

10 +_ 2 3.8 _+ 2.4 5.2 _+ 1.6 8.2 +_ 1.6

10.2 +_ 2.4 4.4 _+ 1.6 5.4 ± 1.4 8.2 _+ 2

12.2 +_ 1.8 4 ± 0.9 5.2 _+ 2 8 _+ 2

12.8 _+ 1.8 4 +_ 2 5 _+ 2 9 ± 2.8

13 ± 2.3 4.2 ___ 1.6 6.8 ± 2.2 9.6 ± 1.4

9.8 ± 2.4 4.4 + 1.4 5.1 ± 1.6 7.6 +_ 1,75

281.5§ ± 8.3 160.6§ ± 11.1 152§ ± 7.3

Nitrogen metabolism in the Indian field crab

227

Table 4. Concentrations (pM.g - l ) of ADP and ATP, and activities ( # M . g - l . h r -1) of ATPase in tissues of O. s e n e x , as for previous tables Time (days), post-operative

Variable (ADP)

Tissue Hepatopancreas Muscle Gill

(ATP)

Hepatopancreas Muscle Gill

ATPase

Hepatopancreas Muscle Gill

0 (unop)

1

2

3

5

7 saline

7 ESE

0.66 _+ 0.14 0.44 _+ 0.06 0.48 _+ 0.02

0.74 + 0.06 0.51 _+ 0.11 0.65* _+ 0.05

0.74 _+ 0.09 0.48 _+ 0.1 0.63* _+ 0.06

0.76 + 0.09 0.47 _+ 0.05 0.65* + 0.06

1.01" + 0.16 0.49 _+0.1 0.71" _+ 0.07

1.09" + 0.17 0.61" + 0.05 0.84* + 0.09

0.69"t" + 0.04 0.48t + 0.02 0.6*t + 0.06

2.32 _+ 0.31 1.85 _+ 0.21 2.06 _+ 0.29

2.2 _+ 0.19 1.68 _+ 0.63 1.85 _+ 0.07

2.17 _+ 0.21 1.68 _+ 0.15 1.88 +_ 0.14

1.89 _+ 0.21 1.61 + 0.1 1.76 _+ 0.1

1.69~: ___ 0.15 1.67 _+ 0.2 1.65 _+ 0.15

1.75~ _+ 0.17 1.57 _+ 0.21 1.66 _+ 0.61

2.05 + 0.19 1.63 ___ 0.15 1.71 + 0.22

1.61 _ 0.17 0.81 + 0.12 1.18 _+ 0.21

1.94 _+ 0.25 1.07 _+ 0.21 1.4 _+ 0.17

1.85 _+ 0.17 0.97 _+ 0.21 1.25 --4-_0.25

1.91 _+ 0.25 1.13 _+ 0.25 1.67" _+ 0.12

2.07* +_ 0.25 1.08 _+ 0.21 1.6 _ 0.25

2.28* -4- 0.35 1.32" _+ 0.27 1.67 _+ 0.3

1.95 + 0.21 1.36" ___0.21 1.36 + 0.21

* > Unoperated control value. "? < Saline control value. :~ < Unoperated control value.

Table 5. Specific activities (nM formazan.mg 1.hr 1) of oxidative enzymes in tissues of O. senex, as for previous tables Time (days), post-operative

Enzyme Succinate dehydrogenase

Tissue Hepatopancreas Muscle Hypodermis Gill

Malate dehydrogenase

Hepatopancreas Muscle Hypodermis Gill

Cytochrome oxidase

Hepatopancreas Muscle Hypodermis Gill

0 (unop)

1

2

111.9 _+ 22.3 112.3 + 22.7 68.7 _+ 13.1 190.6 + 14.2

204.7* _+ 46.8 159.2" + 24.1 117.6* + 28.7 238.2 + 39.6

181.3" ___ 24.1 161.1' + 30.1 98.8* + 11.3 206.3 ___ 37.8

181.9" + 35.9 157.7" ___ 19.7 103.2" + 20.6 222.5 + 44.3

174.5" + 16.7 153.9 + 20.1 101.9" ___ 19 209.1 ___ 38.3

206.6* + 40.9 150" + 14.4 86.3 + 16.2 219.1 _ 43.4

130.5"i" + 9.3 123.9 _+ 12.2 72.6 + 6.8 194.4 + 19.5

38.7 + 12.2 33.5 ___ 14.1 41.1 + 11.4 62.9 _+ 15.5

58.1 + 15.1 51.3 + 13.7 81.7" ___ 19.5 98.6* + 21

57.6 _+ 9.7 44 _+ 7.4 67.5* + 12 92.4 + 16

50.6 + 10.9 43.3 -4- 10.1 68.8* _ 12.8 90 + 13

54.8 + 10.4 46.8 + 14.5 73.3* + 14.8 86.3 + 17.8

58.7 _ 10.5 44.9 +__ 13.5 69.8 + 18.7 84.4 + 15.2

49.1 + 6.1 37.8 ___ 9.7 48.1 + 11.3 63.2 ___ 8.2

2.09 + 0.53 2.11 + 0.38 1.05 + 0.38 3.29 + 0.73

* > Unoperated control value. ~ < Saline control value.

3.09 -+- 0.55 2.8 ___ 0.47 2.05* + 0.47 4.2 + 0.6

3.16" + 0.43 3.14" + 0.55 1.46 ___ 0.24 3.36 + 0.56

3

2.93 + 0.67 3.6* + 0.7 1.29 ___ 0.19 3.39 + 0.69

5

3.36* + 0.27 4.3* + 0.7 1.14 + 0.26 3 + 0.5

7 saline

2.86 + 0.95 3.6* + 0.6 0.96 + 0.27 3.12 + 0.58

7 ESE

2.52 + 0.3 2.7"t + 0.2 0.98' + 0.3 3.15 + 0.82

228

R. RAMAMURTHIet al.

influence on general metabolism. A major question for the future concerns the nature a n d n u m b e r of n e u r o h u m o r s acting. This system, and possibly other crustaceans, should afford a valuable tool for the study of direct control of enzyme activity by endocrine factors. REFERENCES

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