Variations in the body fluid nitrogenous constituents of Cryptochiton stelleri (mollusca) in relation to nutrition and reproduction

Comp. Bzochem. Physml., 1966, Vol. 19, pp. 737 to 744. Pergamon Press Ltd. Printed ~n Great Britain

VARIATIONS IN T H E BODY F L U I D N I T R O G E N O U S C O N S T I T U E N T S OF C R Y P T O C H I T O N S T E L L E R I (MOLLUSCA) IN RELATION TO N U T R I T I O N AND REPRODUCTION * B. S. V A S U t and A. C. GIESE Department of Biological Sciences, Stanford Universaty, Stanford, California, U.S.A. (Received 17 May 1966) Abstract--1. Levels of nitrogenous constituents in Cryptochiton stelleri are

higher than in Pisaster ochraceus and show a relation to the annual reproductive cycle. 2. Starvation resulted in slight decreases in protein and showed no increase in gonad size. 3. Feeding resulted in an initial decrease in protein. There were no increases m gonad size comparable to that seen in field animals.

INTRODUCTION THE importance of blood and body fluids in the maintenance of life and activities of cells is well known. An important constituent of blood involved in such functions is the protein. While lower groups like the echinoderms have low levels of protein in their body fluid (Vasu & Giese, 1966), the higher animals possess body fluids, complex in their composition and including many organic constituents (see Engle & Woods, 1960; Altman, 1961). Several studies have been made on the blood proteins of vertebrates, especially man, because of their greater clinical and economic importance. However, a knowledge of the proteins of invertebrate groups is limited to a few isolated studies and data are scanty and imperfect even today, except perhaps for the insects. The present paper is a study of the protein and non-protein nitrogenous substances in the body fluid of the chiton, Cryptochiton stelleri. In contrast to the sea star, Pisaster ochraceus (Vasu & Giese, 1966), the chiton has a relatively high concentration of nitrogenous substances (Myers, 1920; Tucker & Giese, 1962). A study of the different nitrogenous constituents and their variations during an entire annual reproductive cycle, and after starvation and feeding, is reported here. * This investigation was supported (in part) by Public Health Service Training Grant GM-647 from the National Institute of General Medical Sciences. t Postdoctoral Fellow, Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556. 737

B. S. VASU AND A. C. GIESE

738

MATERIALS AND M E T H O D S T h e chitons were collected f r o m Pigeon Point (south of San Francisco) on the California coast. T h e body fluid was obtained by making a small incision on the ventral side between the foot and ctenidia and allowing the fluid to drain into a beaker. T h e methods used in the determination of total nitrogen, non-protein nitrogen and protein have been described earlier (Vasu & Giese, 1966). Amino acid nitrogen was determined by the ninhydrin method (Spies, 1957). T h e determination of urea nitrogen was based on the method described in Levenson & M c F a t e (1962). T h e urea is hydrolysed to a m m o n i u m carbonate by the enzyme urease. T h e a m m o n i a nitrogen was then determined b y Nesslerization. Uric acid was determined by the colorimetric method based on the color developed by reaction of phosphotungstate reagent with uric acid. T o correct for non-uric acid color, an aliquot of the same sample of body fluid was preincubated with the enzyme uricase which specifically destroys uric acid by converting it to allantoin. T h e difference in color before and after uricase treatment was taken as a measure of the uric acid. T h e details of the method are described in Sigma Tech. Bull.,

No. 680 (1963). EXPERIMENTAL RESULTS

1. Levels of nitrogenous constituents--Table 1 Total nitrogen ( T N ) ranged f r o m 26.6 to 364.7 m g per 100 ml (mg per cent), the higher values occurring during spring, s u m m e r and fall, and the lower values during winter. N o n - p r o t e i n nitrogen ( N P N ) levels were low in spring (April, 3.9 m g per cent) and increased gradually, being highest during winter (February, 11.4 m g per cent). Since N P N is only a small proportion of the T N and is actually highest when T N is lowest, it follows that the fall in T N levels during winter is due to a corresponding fall in protein nitrogen. T h u s the protein values as obtained by b o t h methods (Lowry and micro-Kjeldahl) indicated the same general trends TABLE 1--MEAN LEVELSOF NITROGENOUSCONSTITUENTSIN THEBODYFLUIDOF C. stellerz DURING1963--64

Months Mar. 1963 Apr. May June July Aug. Sept. Oct. Nov. Dec. Jan. 1964 Feb.

No. of Protein Amino acid in&TN NPN (g/100 ml, N AAN Urea N wduals (mg/100 ml) (mg/100 ml) Lowry method) (mg/100 ml) ~ × 100 (rag/100 ml) 9 10 I0 10 10 10 10 10 10 10 9 10

230"6±25 236.6±46 236-7±41 222"8±40 229"3±33 238"4±47 240.8±36 277-1±43 234"4±58 250"1±28 115.9±53 128'9±54

7'0±0-7 3'9±2"5 4-7±1"5 51±0'7 6-3±1"2 5.9±0'6 80±0-9 85±1-4 8"3±1"5 9"8±1"7 9'3±1-0 114±2-2

1'07±0"1 1'24±04 1"56±0"3 1"34±0'2 154±0-4 1"56±0'3 1.31±0"2 1"80±03 1"47±0-5 1"13±0'I 0'67±0"2 0.73±0'4

-1'8±05 -2-5±0"4 2-4±0"4 2-5±0'5 3"3±0"5 3"5±0"7 2'1±0-6 -2"5±06 2-4±0"4

-45 -50 39 42 41 41 26 -27 21

----------9"5 9"0

BODY FLUID

NITROGENOUS

CONSTITUENTS

739

OF CRYPTOCHITON STELLERI

during the course of the year, the higher proportion of the range occurring during spring, summer and fall, and low levels occurring in winter. It was of interest also to determine fractions of the NPN increasing during winter (Table 1). Amino acid nitrogen levels during the year ranged from 1.1 to 5.4 mg per cent. During spring, summer and fall, the proportion of amino nitrogen to NPN is higher (39-50 per cent) while during winter the proportion decreases (21-29 per cent). This indicates an increase in the level of other NPN constituents during the winter months. Analysis for urea and uric acid during winter showed high levels of urea, while no uric acid could be detected by the methods employed. 2. Nitrogenous constituents in relation to nutrition and reproduction A distinct annual reproductive cycle has been described in C. stelleri (Tucker & Giese, 1962), the gonad index being maximum in late winter or early spring, at which time occurs a spawn-out (Fig. 1). In correlation with this cycle is the decline in protein and increase in NPN in winter, observed in the present study. 25

H 0""0 .'*2-

g

Protein

Gonad index

/

20

o

Q /

/

-

6

15

4 0

10

%

05

.......0 ........ 0"'"'0 0

•

M

•

A

2

.-

•

M

....... 0

•

J

I

d 1963

II

A

•

S

0

m

N

•

D

•

J

C)

F 1964

FIG. 1. Monthly levels of body fluid protein and mean values of gonad index in C. stelleri during 1963-64. The fall in protein in winter may be related to the demands for nutrients by the rapidly developing gonad. The fall in amino acid nitrogen, like the fall in protein, may also be related to the nutritional requirements of the growing gonad. However, the algae on which the chitons feed are less plentiful in winter and differ qualitatively with season (Black, 1950; Channing & Young, 1952, 1953; Wart, 1955; Jones, 1957), so that the availability of nutrients may also influence protein and amino acid nitrogen levels. The increase in NPN levels (especially urea) may indicate utilization of protein either from the body fluid or from extravascular sources during such periods of inanition. 25

740

B.S. Vhsu ANDA. C. GIESE

In order to determine the relation between gonadal growth, nutritional factors and body fluid protein levels, changes in gonad index and levels of nitrogenous constituents were followed in two starvation experiments.

Starvation experiments Experiment 1. Chitons were collected in the fall, when protein levels are high and gonad index small. These were starved through winter and early spring for a total period of 140 days. The body fluids were sampled after 53 and 93 days of starvation so that it was possible to obtain data on levels of nitrogenous constituents in the body fluids of the same individuals, without killing them. The determination of their gonad index, however, required their sacrifice. This was done 140 days after starvation. However, a few chitons were sacrificed at 93 days to determine the gonad index at this stage of starvation. The data are presented in Table 2 and Fig. 2, and include values from corresponding monthly field collections. T A B L E 2 - - L E V E L S OF NITROGENOUS CONSTITUENTS I N THE BODY FLUID OF

C. stelleri.

(Starvation from 30 November 1963 through 17 April 1964) 20 Jan. (53 days)

1 March (93 days)

17 April (140 days)

--

3"0 ± 1'5

2"7 ± 1"7

5.7±1.6

8.8±2.4

2.6±1-0

Starved

294"7± 51"7

200"2± 35'9

179"2± 20-9

F~eld

115.9±52.8

128"9±54"5 166"8±52"3

Starved

68-2 ± 10'3

30 Nov. Starved Gonad index

4"2 ± 1'2 Field

Total Nitrogen (rag/100 ml) Non-protein nitrogen (mg/100 ml)

234.4 ± 57.5 11"4 + 3'7

6"6 ± 1"4

9'3±1'0

11"4±2"2

8'6±1"3

Starved

1 31 ± 0 " 3

1"20±0"2

1'08±0"3

Fteld

0"67±0"2

0"73±0'4

0'99±0-3

8"3 ± 1"5 F~eld

Protein (g/100 ml) Ammo acid nitrogen (mg/100 ml)

1-5 ± 0"5 Starved

--

1'50 ± 0"993

3'2 ± 0-6

2'14 + 0-6 Field

2"5±0"6

2.4±0.4

2-2±0.9

It is evident from the data that with starvation there is a retardation m the development of the gonads and only small decreases in levels of protein. Since the decrease in body fluid proteins of field animals during the corresponding period is more remarkable, and takes place with simultaneous increase in gonad size, the fall in levels in field animals appears to be related to the increase in size of the gonads.

BODY FLUID NITROGF_~/OUS CONSTITUENTS OF C R Y P T O C H I T O N S T E L L E R I

741

The remarkable increase in N P N at the end of the first period of starvation suggests the utilization of protein as a source of energy. Since the fall in body fluid protein does not account for the increase in NPN, extravascular sources are probably involved. The decrease in level of N P N towards the later stages suggests a regulation. As adequate amounts of the body fluid were not available, analyses for fractions of N P N increasing with starvation could not be made. However amino acid nitrogen levels showed wide individual variations suggesting their utilization and maintenance of their supply by proteolysis of tissue proteins. Starved ..•..F=eld StarvedFleld Gonad index 1

I J

20

B

15

l'O

0.5

0~

N

D 1964

d

F

M

A

1965

FIG. 2. Effects of starvation from November 1963 to April 1964 on levels of body fluid protein and gonad index in C. stelleri. The gonad index are from Lawrence et al. (1965).

Experiment 2. In this experiment the effects of starvation were studied at a time when the reproductive stress is minimal. Thus chitons were collected earlier in the year, i.e. in the late spring, when the gonad index is low and growth of gonad is also slow but protein level high. These were first starved for a period of about 3 months and then divided into two groups. One group was placed in a tank to which large quantities of algae lridaea, Macrocystis and Gigartina-were supplied, while the other group was placed in a tank to which no algae or other food had been added. The body fluids of individuals of both batches were sampled after 171 days and finally sacrificed after 238 days. Thus it was possible to compare the effects of feeding and starvation on the levels of body fluid nitrogenous substances simultaneously. The data are presented in Table 3. It is evident from the data obtained in both experiments that starvation both during spring and summer months (when there is no increase in gonad size) or during winter (when there is an increase in size of gonad) does not bring about decreases in body fluid protein levels comparable to that obtained in field animals during winter. In chitons supplied with food, on the other hand, there is a decrease

742

B. S. VASU AND A . C. GIESE

in level of protein. However, the gonad index of the chitons at the end of the first stage of experimentation could not be determined as the animals were not sacrificed and only body fluids were sampled. But the gonad index at the end of the experimental period was still low in animals supplied with food, though higher than in the starved chitons. Neither was any spawning observed in the tanks in which these chitons were maintained. It appears therefore that in animals maintained in the laboratory (either with or without food) no increases in gonad size occur comparable to that seen in field animals. Other factors, external or internal, or a combination of both are probably necessary for normal growth and development of the gonads (Tucker & Giese, 1962). T A B L E 3 - - L E V E L S OF NITROGENOUS CONSTITUENTS I N THE BODY FLUID OF

C. stellert.

(Feeding and starvation from 16 May 1964-9 January 1965) Date of collection: 16 May 1964

Levels in May 1964

Starved to 10 Aug. 1964 (87 days)

Protein (g/100 ml)

1"55 + 0-3

1'4 + 0"2

Non-protein nitrogen (mg/100 ml) Amino acid nitrogen (mg/100 ml)

Gonad index

4"65 ± 1-5

1"75 + 0'5

To 4 Nov. 1964 (171 days)

To 9 Jan. 1965 (238 days)

Fed

0"57 _+0-24

0"56+ 0-24

Starved

097+05

0-98_+0"12

Fed

7"8 + 0"8

6"4 +_0-4

Starved

7'7 _+1-5

6'2 _+0-0

Fed

0'6 + 0"1

1"4 + 0"5

Starved

1"4 + 0"1

1-5 + 0-3

Fed

--

0 4 + 0-1

Starved

--

0'6 _+0"3

6"9 +_0'7

1"9 + 0'2

0-96

In both experiments, starvation does not bring about as remarkable a decrease in body fluid protein as in the case of fed or field animals. It appears therefore that the body fluid protein is not a major source of nutrients during starvation. T h e increase in N P N with starvation indicates utilization of protein, probably from extravascular sources. T h e levels of amino acid nitrogen show wide variations, both after feeding and after starvation, some individuals having high levels and others low. This probably indicates their probable utilization and maintenance of their supply by proteolysis of tissue proteins. Further studies on the relation of amino acids to the nutritional and reproductive physiology of the animals are indicated. DISCUSSION T h e body fluid of C. stelleri contains a higher level of protein than Pisaster ochraceus. During the year values ranging from 0.16 to 3.5 g per cent were

BODY FLUID NITROGENOUS CONSTITLrENTS OF C R Y P T O C H I T O N S T E L L E R I

743

determined by the Lowry method. Though a broad range of levels does occur in the chitons, the variation is not as wide as that described for P. ochraceus (Vasu & Giese, 1966) or for arthropod species (Leone, 1953). It has been stated that the protein content of the blood is incontestably corrclated with the position occupied by the organism in the zoological scale, the highest levels being found in the most highly evolved groups (Florkin, 1949). However, extremes of levels may occur in members of a single phylum or individuals of a single species. Such variations may depend upon the season, temperature, sex, size, age, physiological state, etc., of the animals, so that values of a few individuals mean little unless the condition of the animal is stated (Prosser & Brown, 1961). It is evident, however, that the increase in levels of protein from the body fluids of simple forms like P. ochraceus (Echinodermata) to the hemolymph of forms like C. stelleri is remarkable, indicating two distinct levels of organization. Thus the body fluids of P. ochraceus show wide variations in the levels of protein and other nitrogenous and organic substances among individuals of a single collection, though all individuals were taken from a restricted area, and were similar in size and reproductive state. No seasonal variations in levels or variations in relation to diffcrcnt factors such as degree of gonadal development or nutritional state were observed. No clear-cut correlation was observed between starvation and feeding for short periods of time and protein and N P N content, both high and low levels occurring in starved and fed forms (Vasu & Giese, 1966). In C. stelleri, on the other hand, distinct seasonal variations occur in levels of protein and other N P N substances in the body fluids. Starvation and feeding experiments have also indicated the relation between levels of nitrogenous substances and growth of gonad. However, the decrease in body fluid protein during starvation does not correspond to the increase in N P N levels, so that extravascular sources are probably involved (see Wigglesworth, 1934). The later decreases in N P N levels suggests a regulation. A loose regulation of N P N levels has been observed in several species of echinoderms (Bennett & Giese, 1955; Tanaka, 1958; Ferguson, 1964; Vasu & Giese, 1966). However, it is obvious that variations and regulations of nitrogenous substances follow more distinct and characteristic patterns in C. stelleri than in P. ochraceus. Acknowledgements--We wish to acknowledge the advice and assistance provided by Drs. H. S. Loring, J. H. Phillips, John S. Pearse and J. M. Lawrence during the course of this investigation. B. S. V. also acknowledges his debt to the United States Educational Foundations in India for the award of a Fulbright grant and the Department of Biological Sciences, Stanford University, for the award of the George Van Vleet, Jr. Memorial Fellowship, 1965.

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B. S. VAsu AND A. C. GIESE

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