Hemolymph-free amino acids and related nitrogenous compounds of Crassostrea virginica infected with Bucephalus sp. and Minchinia nelsoni

Comp. Biochem. Physiol., 1970, Vol. 34, pp. 547 to 556. PergamonPress. PHntedin Great Britain

HEMOLYMPH-FREE AMINO ACIDS A N D RELATED NITROGENOUS COMPOUNDS OF C R A S S O S T R E A V I R G I N I C A INFECTED W I T H B U C E P H A L U S SP. AND MINCHINIA NELSONI* S. Y. F E N G , E. A. K H A I R A L L A H and W. J. C A N Z O N I E R Marine Research Laboratory and Biological Sciences Group, University of Connecticut, Noank, Connecticut 06340; Biochemistry and Biophysics Section, Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268; and N. J. Oyster Research Laboratory, Rutgers, The State University, New Brunswick, New Jersey 08903 (Received 17 October 1969)

Abstract--1. Seventeen free amino acids and related amines were detected in the normal oyster hemolymph. Free taurine, alanine, glycine and serine were found in high concentrations. Phosphoethanolamine, fl-alanine, ornithine, phosphoserine, arginine, lysine and glutamic acid were present in moderate amounts, while leucine, isoleucine, y-aminobutyric acid, aspartic acid, threonine and methionine existed in low and trace quantities. 2. The total concentration as well as composition of normal oyster hemolymph-free amino acids and amines varied with seasons and geographic locations at which the oysters were maintained. 3. In general the depletion of free alanine, glycine, serine, phosphoethanolamine, fl-alanine, omithine, arginine, lysine, leucine and isoleueine was accompanied by concomitant increases in free taurine, aspartic acid, glutamic acid, threonine and phosphoserine in the infected oyster hemolymph.

INTRODUCTION HISTOPATHOLOGY of oysters infected with Bucephalus sp. (Cheng & Burton, 1965) and Minchinia nelsoni (Farley, 1965; Haskin et al., 1965; Haskin et al., 1966) has been well documented. Recently Feng & Canzonier (1970) have reported changes in h e m o l y m p h proteins of oysters infected with these parasites. Although there have been several studies dealing with the free amino acid composition of molluscan adductor muscle as related to external osmotic concentrations (Bricteux-Gr6goire et al., 1964a, b, c; L y n c h & Wood, 1966), only Muller (1963), to the best of our knowledge, has attempted to determine the hemolymph amino acid content of Crassostrea virginica. Since the oysters used in Muller's study were from Delaware Bay and no effort was made to determine the degree of M . nelsoni infection in them, * Contribution No. 63 from Marine Research Laboratory, University of Connecticut supported, in part by a Biomedical Sciences Support Grant FR-07014 from the National Institutes of Health to the University of Connecticut and by a contract No. 14-17-0007-887 from the Bureau of Commercial Fisheries to Dr. H. H. Haskin, N.J. Oyster Research Laboratory, Rutgers, The State University. 547

548

S. Y. FENG, E. A. KHAIRALLAHAND W. J. CANZONIER

his result on the hemolymph amino acid content represents only a composite of normal and diseased oysters. This paper reports a preliminary account of variation of oyster h e m o l y m p h nonprotein nitrogenous compounds associated with geographic locations, seasons and parasitic infections. MATERIALS AND METHODS Oysters from the Navesink River, New Jersey were exposed to the infection of M. nelsoni by transplanting them to Delaware Bay, an endemic area of the disease. Concurrently another group of the same oysters was moved to Monmouth Beach, New Jersey, a nonendemic area of M. nelsoni. On the average about one-third of the Navesink River oysters were infected with the trematode, Bucephalus sp. The transplants were sampled during the winter and summer after a 9 to 12 months' residence in their respective sites. The animals were exsanguinated via the adductor muscle for the determination of hemolymph nonprotein nitrogenous compounds. Hemolymph samples, following the removal of cellular elements by centrifugation, were stored at - 2 0 ° C until used. Histological techniques were used to determine the degree of the infections in the host tissues. The exsanguinated oysters were fixed in Davidson's fixative. The specimens were processed and stained in hematoxylin and eosin according to established histological procedures. Sections were then examined for evidence of any recognizable pathology, general tissue conditions and the presence of the parasites. Based on histopathologic examinations, oysters were divided into four groups: the normals, the Bucephalus-infected, the Minchinia-infected and the Bucephahts-Minchinia-infected. Uninfected oysters in both locations served as controls. Hemolymph samples were pooled according to the four categories. The pooled hemolyph samples were deproteinized in 10% trichloroacetic acid and ultrafiltered through a 100~k Millipore filter. Aliquots of the ultrafiltrates were analyzed on a Beckman Model 120C amino acid analyzer using the expanded scale. RESULTS

Nonprotein nitrogenous constituents of oyster hemolymph T h e oyster h e m o l y m p h nonprotein nitrogenous compounds consist of three major constituents which are urea, ammonia and amino acids (Table 1). I n general, the first two components make up about 52 to 79 per cent of the total nonprotein nitrogen in summer and 16 to 47 per cent in winter normal oysters. Urea contents, with two exceptions, are always higher than ammonia. According to H a m m e n et al. (1966), the excretion products of oysters consisted of 65 to 68 per cent ammonia, 8 to 13 per cent urea and 5 to 20 per cent amino acids; they concluded that C. virginica is ammontelic as are other marine molluscs. I n the present study, relatively low concentrations of ammonia in hemolymph as contrasted with those of urea could be attributed to the higher excretion rates of the ammonia than those of the urea. Seventeen free amino acids and related amines were detected in the normal oyster hemolymph. These can be grouped into three categories according to their concentrations. Taurine, alanine, glycine and serine are found in high concentrations. Phosphoethanolamine, fl-alanine, ornithine, phosphoserine, arginine,

549

HEMOLYMPH-FREE AMINO ACIDS IN INFECTED OYSTERS lysine and glutamic acid are present in moderate leucine, y-aminobutyric

amounts,

while leucine, iso-

acid, aspartic acid, threonine and methionine exist in low

and trace quantities. Histidine, proline, cystine, valine, tyrosine and phenylalanine were not detected in any samples. TABLE 1 .--FREE AMINO ACIDS AND RELATED COMPOUNDS IN THE POOLED HEMOLYPH SAlx.IPLES OF NORMAL AND INFECTED OYSTERS (EXPRESSED AS n m o l e s / m l HEMOLYMPH) Summer Monmouth Beach

Cape Shore Amino acid or compound

NOR

BUC M I N

Winter

MINBUC

NOR

Monmouth Beach

Cape Shore

BUC N O R

BUC

MIN

MINBUC

NOR

BUC

Ammonia

725 520

488 356

460 219

714 368

1980 512

624 449

-299

440 344

438 589

352 85

1030 399

318 128

Tau Ala Gly Ser

250 229 124 120

58 56 46 14

154 168 56 26

261" 171 56 16

152 104 106 53

324* 70 85 26

112 330 109 87

354* 119 39 74

189" 219 108 93*

314" 89 61 20

68 263 136 339

376* 261 118 182

PE Bala Om PS Arg Lys Glu

72 71 69 62 27 20 12

19 60 25 200* 8 + 8

15 18 30 ---33*

75 28 43 21 -+ 103"

57 43 14 76 17 7 32

46 14 19" 112" --22

434 104 111 142 24 17 114

191 35 54 5 16 9 46

588* 74 53 10 12 43* 14

136 27 71 72 -11 30

416 68 97 101 24 23 57

167 55 44 131" 21 19 59

Leu lleu GABA Asp Thr Met

8 3 3 + + --

5 + + 11" 5* +

--8* 32* 4* --

--21" 37* 6* --

Urea

Total amino acids No. of oysters

7 + 2 + + +

6

5

--

+

--

÷

+

5*

6

+

+

--

-t-

+

+ 15" 5* 8*

11 7 + +

46* 32* ---

10 2 -+

1021

1418

676

1631

1462

7

4

11

5

1172

519

544

839

674

758

1614

3

2

13

3

13

7

3

3

9 35* + --

33 3 1 --

19 7* + --

NOR, normal; BUC, Bucephalus-infected; M I N , Minchinia-infected; M I N - B U C , infected with both parasites; GABA, 7-aminobutyric acid; PE, phosphoethanolamine; PS, phosphoserine; + , trace quantities or represents less than 1 nmole/ml hemolymph. In calculating the total amino acids, however, + is counted as 1 nmole; * denotes increases in amino acid and amine concentrations of infected oysters as contrasted with their appropriate controls (NOR). C h a n g e s i n t h e t o t a l f r e e a m i n o a c i d s i n t h e n o r m a l vs. i n f e c t e d o y s t e r s a r e r e a d i l y d i s c e r n i b l e i n T a b l e 1. T h e d a t a e x t r a c t e d f r o m T a b l e 1, a r e g r a p h e d a n d s h o w n i n F i g . 1. A t C a p e S h o r e , r e g a r d l e s s o f s e a s o n s , t h e t o t a l f r e e a m i n o a c i d s of t h e n o r m a l or u n i n f e c t e d oysters are c o n s i s t e n t l y h i g h e r t h a n t h o s e of t h e

550

S.Y. FENG, E. A. KHAIRALLAHAND W. J. CANZONIER

infected ones. The differences in the total free amino acids between the normal and infected reach a minimum during the winter season. This observation probably reflects the relative quiescence of the parasites. Lowering of ambient temperatures may conceivably affect the rate of transport and utilization of host-free amino acids by the parasites. TOTAL FREE AMINO ACIDS CAPE SHORE

1.8'

MONMOUTH BEACH

[

1.6 I

1.4'

m

1.2, O

.c 1.0, .8'

::::::::::::

>::::::::::

~..4 .2

:i:!$1:i:!

W

.,, .,.,.

Hw

Rw

::5::Z::

Norrna

Minchinia

B ucephalus

Min.-Buc.

Norma

V

Bucephalus

FIG. 1. Seasonal and geographic variations in the total concentration of free amino acids and amines in the normal, Minchinia-,Bucephalus-and Minchinia-Bucephalusinfected oyster hemolymph. "S" and "W" represent summer and winter concentrations respectively.

At Monmouth Beach, differences in the total free amino acids between the normal and infected oysters are probably not significant; however, it should be pointed out here that the shift in amino acid composition is still apparent. Therefore, the use of total free amino acid as an indicator for distinguishing the normal from diseased oysters has its limitations. The data strongly suggest seasonal variations in the total free amino acid content in nearly every group examined, the only exception is in the oysters infected with both Minchinia and Bucephalus where the observed difference between summer and winter animals is probably not significant (Fig. 1).

Variations in the normal oyster hemolymph free amino acids Cognizance of the variation in the normal hemolymph-free amino acid composition is a prerequisite to determine the metabolic disorder of amino acids in the infected host. Figure 2 depicts seasonal and geographic variations in the normal oyster hemolymph-free amino acids. Taurine decreases in its concentration during the winter while alanine and fl-alanine show increases. Similar seasonal variations are detected in leucine, serine, phosphoethanolamine, ornithine, phosphoserine, glutamic acid, y-aminobutyric acid and aspartic acid. These changes are consistent in the two locations, Cape Shore and Monmouth Beach.

551

HEMOLYMPH-FREE AMINO ACIDS IN INFECTED OYSTERS

Geographic variations in the concentrations of certain amino acids are also apparent in Fig. 2. When one compares the concentrations of taurine, alanine and fl-alanine in oysters held at Cape Shore with those maintained at Monmouth Beach during summer and winter, the concentrations of these amino acids are always higher in Cape Shore transplants than those held in Monmouth Beach waters. These differences to a large extent could be ascribed to the salinity regime of the two areas; the salinity of Cape Shore is higher than that of the Monmouth Beach. It is known that the increase in adductor muscle taurine and alanine concentrations can be correlated with increasing ambient salinities (Lynch & Wood, 1966). .c 0.4[

Normal oysters Capeshore

Monmouth beach

V-!

I

I I r-'q

J l

•

,

I I

~ 0.2 E

ii r~.-1

I

C Taurine

.I

Alonine

[--q i w'

,8-olanine

Taurine

Alonine

Fzc. 2. Seasonal and geographic variations in the concentration alanine and/3-alanine in the normal oyster hemolymph. "S" and summer and winter concentrations respectively.

,g- olanine of free taurine, "W" represent

Shift in hemolymph free amino acids associated with infections Having recognized variations in free amino acids of normal oysters attributable to seasons and geographic locations, the shift in free amino acid pools associated with parasitic infections can be readily ascertained. In Table 1, figures that are starred denote increases in amino acid and amine concentrations of infected oysters as contrasted with their appropriate controls or uninfected oysters• It is obvious that there are more amino acids being depleted than increased. In the infected oysters, considerable loss of amino acids is encountered in those amino acids present in high and moderate concentrations, while increases of amino acids are observed in those normally present in low or trace quantities. In general, with a few minor exceptions, infected oysters hemolymph samples show substantial reductions in the concentration of alanine, glycine, serine, phosphoethanolamine,/%alanine, ornithine, arginine, lysine, leucine and isoleucine. Considerable increases in aspartic acid are observed in all infected oysters except winter Minchinia-infected oysters at Cape Shore. The concentrations of taurine are reduced to approximately a quarter to one-half of the normals in summer

552

S.Y. FENG, E. A. KHAIRALLAHAND W. J. CANZONIER

Bucephalus- and Minchinia-infected oysters at Cape Shore, while the other six groups of infected oysters exhibit definite increases in taurine concentrations. Aspartic acid became more concentrated in seven out of eight infected groups and less concentrated in winter Cape Shore Minchinia-infected oysters as contrasted with their respective controls. Augmentations of threonine concentration occurred only in summer infected oysters. Summer Cape Shore Minchinia-infected and Minchinia-Bucephalus-infected oysters were the only groups which showed increases in glutamic acid. Increases in phosphoserine are probably associated with Bucephalus sp. infection.

Miscellaneous observations Methionine was either in trace quantities or totally missing from the hemolymph. No free aromatic amino acids were detected in the normal and infected oysters. Phosphoethanolamine, phosphoserine and ~,-aminobutyric acid were detected for the first time in oyster hemolymph. Since phospholipids have been demonstrated in scallops (Shieh, 1968) it is reasonable to assume that phosphoethanolamine and phosphoserine are associated at least in part with the metabolism of phospholipids in the oyster. The presence of ~,-aminobutyric acid, a naturally occurring amine of nonprotein derivation, is of some interest, because it is found in higher plants (Thompson et al., 1953), certain bacteria, fungi (Holden, 1962) and nervous tissue of vertebrates and invertebrates. Its inhibitory effect on the crayfish stretch receptor has been well documented (Bazemore et al., 1956; also see review by Tallan, 1962). The functional significance of this amino acid in oysters has yet to be determined. DISCUSSION The reduction of hemolymph-free amino acids in infected oysters could be attributed to the presence of the parasites, while the augmentation of certain amino acids might be interpreted as the host's effort to replenish those lost to the parasites and/or as being contributed by the parasites. To answer these questions, one needs to know more about the nutritional requirement and intermediary metabolism of the host and parasites. The depletion of alanine appears to be linked to the concomitant increase in aspartic acid and to some extent glutamic acid (Table 2). Via oxidative deamination the aspartic acid could be converted to oxaloacetate which in turn was decarboxylated to pyruvate. Thus, alanine may be synthesized from pyruvate and glutamic acid by transamination. Hammen (1968) found that oysters exhibited a very low rate of production of pyruvate from alanine, suggesting an alanine aminotransferase more efficient in the direction of alanine production. He further demonstrated that rates of amino acid excretion and activities of aminotransferase in tissues were closely correlated, which suggests that aminotransferase is important in replacing amino acids lost to the medium. Such a mechanism might be employed to replenish the depleted amino acids in parasitized oysters.

HEMOLYMPH-FREE AMINO ACIDS IN INFECTED OYSTEI~

553

TABLE 2--CoMPF_a'qSATORYCHANCESIN TAUaINE(Tau), ALANr_WR(Ala), nsPnaTiC (Asp) AND GLUTAMIC ACID (Glu) IN HEMOLYMPH OF SOME INFECTED OYSTERS Oysters infected with Amino acid

BUC

MIN

MIN-BUC

Tau Ala Asp Glu Tau Ala Asp Glu

23* 24 110 67 213 67 150 69

62 73 320 275

104 74 370 858

Tau Ala Asp Glu Tau Ala Asp Glu

316 36 457 40 553 99 104 233

169 66 29 12

280 26 500 27

Location

Season

,Cape Shore

Summer

,Monmouth Beach

Summer

,Cape Shore

Winter

,Monmouth Beach

Winter

* As per cent of the normals. BUC, Bucephalus-infected; MIN, Minchinia-infected; MIN-BUC, infected with both. Mengebier & Wood (1969) reported that phosphohexose isomerase activity in

M. nelsoni infected oyster hemolymph was greatly reduced; the normal glycolysis and glycogen synthesis could be blocked since this enzyme catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate. This blockage at the beginning portion of the glycolytic sequence could conceivably lead to the depletion of pyruvate and eventually impair the pathway by which alanine is synthesized by transamination. Thus, decreases of alanine in the infected host in our study may be explained in part by this mechanism. The pyruvate pool, however, could be resupplied directly from oxaloacetate derived from aspartic acid by the action of pyruvic carboxylase, or indirectly by decarboxylation of oxaloacetate to phosphoenolpyruvate (phosphoenolpyruvic carboxykinase) and then to pyruvate. Jodrey & Wilbur (1955) found pyruvic carboxylase to be present in high concentrations in the oyster mantle, while phosphoenolpyruvic carboxykinase was also present but in low concentrations (Simpson & Awapara, 1966). In short the formation of a-ketoglutarate and oxaloacetate from glutamic and aspartic acid respectively provided not only pathways for the conversion of the keto acids to amino acids by transamination but also furnished akernative sources for replenishing the pyruvate pool, a direct breakdown product of glucose. Phosphoserine could serve as a source of serine lost in oysters infected with Bucephalus sp. (Table 3). This reaction is catalyzed by phosphatase. It is also

554

S. Y.

FENG,

E. A. KHAIRALLAHANDW. J. CANZONIER

known that there is a rapid interconversion of serine and glycine. Thus, glycine could also be replenished in this manner. However, the accumulation of phosphoserine may be interpreted as evidence of failure to convert this amino acid to serine due to the lack of phosphatase. Eble (1966) reported a significant decrease of tissue acid phosphatase in oysters heavily infected with M. nelsoni. Hence, similar alternative interpretations could be applied to the increase of aspartic acid, threonine and glutamic acid.

(Gly), SERINE (Ser) AND (Ps) IN Bucephalus sP. INFECTEDOYSTERHEMOLYMPH

TABLE 3--COMPENSATORY CHANGES IN GLYCINE

Amino acid

Oysters infected with Bucephalussp.

Gly Ser Ps Gly Ser Ps

37* 12 322 80 49 147

Gly Ser Ps Gly Ser Ps

36 85 35 87 54 130

Location

PHOSPHOSERINE

Season

t Cape Shore

Summer

.~Monmouth Beach

Summer

l Cape Shore

Winter

}-Monmouth Beach

Winter

J

* As per cent of the normals. Noteworthy are increases of taurine in the infected oysters with the exceptions of those infected with Bucephalus sp. and M. nelsoni in summer at Cape Shore. Such increases in the taurine concentration could provide for the maintenance of the internal osmotic concentration which was lowered due to the depletion of free amino acids in the infected host. The role of free amino acids in maintaining the osmotic balance of oysters has been convincingly demonstrated by Lynch & Wood (1966); Hammen (1969) recalculated their data and found that the free amino acids accounted for about 13.9 to 21.0 per cent of the total of osmotically active substances in oysters. The depletion of arginine in infected oysters may have serious consequences in those metabolic pathways requiring ATP, since arginine is the basis of the phosphagen phosphoarginine in many invertebrates. Arginine may be converted to ornithine and urea by the enzyme, arginase which is known to be present in the oyster (HammeD et al., 1962; Gaston & Campbell, 1966). The increase in urea may be due to the decrease in arginine.

HEMOLYMPH-FREE AMINO ACIDS IN INFECTED OYSTERS

555

In conclusion, we feel that the shift in the free amino acid pool in infected oysters is not fortuitous; it probably reflects the host's effort to replenish the depleted amino acids and maintain the osmotic concentration of the host's "milieu int6rieur". These compensatory responses are achieved by the conversion of keto acids to amino acids and by increasing the taurine concentration via some unknown pathways. SUMMARY I. Analyses of nonprotein nitrogenous compounds in the hemolymph of oysters infected with Bucephalus sp. and Minchinia nelsoni have been carried out

by the established liquid chromatographic procedures. 2. With the exception of ammonia and urea, taurine, alanine, glycine and serine are found most abundant in the oyster hemolymph. Phosphoethanolamine, fl-alanine, ornithine, phosphoserine, arginine, lysine and glutamic acid are present in moderate amounts, while leucine, isoleucine, 7-aminobutyric acid, aspartic acid, threonone and methionine exist in low and trace quantities. 3. Variations in the composition of normal oyster hemolymph free amino acids may be attributable to seasonal and geographic locations at which the oysters were maintained. 4. In general, with a few exceptions, infected oyster hemolymph samples show substantial reductions in the concentration of alanine, glycine, serine, phosphoethanolamine, fl-alanine, ornithine, arginine, lysine, leucine and isoleucine. Considerable increases in taurine, aspartic acid, glutamic acid, threonine and phosphoserine are observed in most infected oysters. 5. This shift noted in the free amino acid pool of infected oysters is discussed in relation to the probable disturbances in the host amino acid and carbohydrate intermediary metabolism and the compensatory responses initiated by the host. REFERENCES

BAZEMOREA. W., ELLIOTTK. A. C. & FLOREYE. (1956) Factor I and y-aminobutyric acid. Nature, Lond. 178, 1052-1053. BRICTEUX-GRI~.GOIRES., DUCH.~TEAU-BossoNCH., JEUNIAUXCH. • FLORKINM. (1964a) Constituants osmotiquement actifs des muscles adducteurs de mytilus edulis adapt6e l'eau de mer ou ~ l'eau saum~tre. Archs int. Physiol. Biochem. 72, 116-123. BRICTEUX-GR~cOIRES., DUCHATEAu-BossoNCH., JEUNIAUXCH. & FLORKINM. (1964b) Constituants osmotiquement actif des muscles adducteurs d'Ostrea edulis adapt6e l'eau de mer ou {t l'eau saum~tre. Archs int. Physiol. Biochem. 72, 267-275. BRICTEUX-GRI~GOIRE S., DUCHATEAU-BossoN CH., JEUNIAUXCH. & FLORKINM. (1964c) Constituants osmotiquement actif des muscles adducteurs de Gryphaea angulata adapt6e a l'eau de mer ou ~ l'eau saum~tre. Archs int. Physiol. Biochem. 72, 835-842. CHENGT. C. & BURTONR. W. (1965) Relationships between Bucephalus sp. and Crassostrea virginica: histopathology and sites of infection. Chesapeake Sci. 6, 3-16.

EBLEA. E. (1966) Some observations on the seasonal distribution of selected enzymes in the American oyster as revealed by enzyme histochemistry. Proc. Shellfish Assoc. 56, 37-42. FARLEYC. A. (1965) Pathologic responses of the oyster, Crassostrea virginica (Gmelin), to infection by the protistan parasite MSX. Bull. Am. Malac. Un. 32, 23-24.

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S. Y. FENG,E. A. KHAIRALLAH ANDW. J. CANZONIER

FENg S. Y. & CANZONIER W. J. (1970) Humoral responses in the American oyster (Crassostrea virginica) infected with Bucephalus sp. and &linchinia nelsoni. Am. Fish. Soc., Special Publ. No. 5, Symposium on Diseases of Fish and Shellfish. (In press.) GASTON S. & CAMPBELL J. W. (1966) Distribution of arginase activity in mollusks. Comp. Biochem. Physiol. 17, 259-270. HAMMEN C. S. (1968) Aminotransferase activities and amino acid excretion of bivalve molluscs and branchiopods. Comp. Biochem. Physiol. 26, 697-705. HAMMEN C. S. (1969) Metabolism of the oyster, Crassostrea virginica. Am. Zoologist 9, 309-318. HAMMEN C. S., HaNLON D. P. & LUM S. C. (1962) Oxidative metabolism of Lingula. Comp. Biochem. Physiol. 5, 185-191. HAMMEN C. S., MILLER H. F., JR. & GEER W. H. (1966) Nitrogen excretion of Crassostrea virginica. Comp. Biochem. Physiol. 17, 1199-1200. HASKIN H. H., CANZONIERW. J. & MYHRE J. L. (1965) The history of " M S X " on Delaware Bay oyster grounds. Annual Rep. Am. Malac. Un. 1965, 20-21. HASKIN H. H., STAUBERL. A. & MACKIN J. G. (1966) Minchinia nelsoni n.sp. (Haplosporida, Haplosporidiidae): causative agent of the Delaware Bay oyster epizootic. Science 153, 1414-1416. HOLDEN J. T. (1962) The composition of microbial amino acid pools. In Amino Acid Pools (Edited by HOLI~ER J. T.), pp. 73-108, Elsevier, Amsterdam, London and New York. JODREY L. H. & WILBER K. M. (1955) Studies on shell formation--IV. T h e respiratory metabolism of the oyster mantle. Biol. Bull. 108, 346-358. LYNCH M. P. and WOOD L. (1966) Effects of environmental salinity on free amino acids of Crassostrea virginica Gmelin. Comp. Biochem. Physiol. 19, 783-790. MENaEBIER W. L. and WOOD L. (1969) The effects of Minchinia nelsoni infection on enzyme levels in Crassostrea virginica--II. Serum phosphohexose isomerase. Comp. Biochem. Physiol. 29, 265-270. MULLER E. W. (1963) Studies on the blood of the American oyster and razor clam. B.A. Thesis, Rutgers, T h e State University, New Brunswick, N.J. SHIEH H. S. (1968) T h e characterization and incorporation of radioactive bases into scallop phospholipids. Comp. Biochem. Physiol. 27, 533-541. SIMPSON J. W. & AWAPARAJ. (1966) T h e pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548. TALLnN H. H. (1962) A survey of the amino acids and related compounds in nervous tissue. In Amino Acid Pools (Edited by HOLDER J. T.), pp. 471-485. Elsevier, Amsterdam, London and New York. THOMPSON J. F., POLLARD J. K. & STEWARD F. C. (1953) Investigations of nitrogen compounds and nitrogen metabolism in p l a n t s - - I I I , y-Aminobutyric acid in plant, with special reference to the potato tuber and new procedure for isolating amino acids other than s-amino acids. Plant Physiol. 28, 401--414.

Key Word Index--Free amino acids and related nitrogenous compounds; oyster; hemolymph ; Crassostrea virginica; infected with Bucephalus sp. and Minchinia nelsoni.