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The biochemical composition of the nauplius stages of Tetraclita squamosa rufotincta Pilsbry
J. exp. mar. Biol. Ecol., 1981, Vol. 51, 0 Elsevier/North-Holland Biomedical
THE BIOCHEMICAL
pp. 241-246 Press
COMPOSITION
OF TETRACLITA
Y. Department
OF THE NAUPLIUS
SQUAMOSA
of Life Sciences,
RUFOTINCTA
STAGES
Pilsbry
ACHITUV Bar-Ilan
University,
Ramat-Gan,
Israel
Abstract: The biochemical composition of laboratory-reared nauplius stages of Tetraclita squamosa rufotincta Pilsbry during development is described. The larvae lose weight progressively during development. Proteins contribute most of the energy during development and it is suggested that protein serves as the source of nitrogen and glucose for the formation of chitin. Lipids contribute some energy during the initial stages of development, and later may serve as an energy source for the cyprids. The rise in RNA values is related to the increase in protein metabolism, particularly in the later stages. DNA values increase only during the early stages suggesting that later development is concerned mainly with organization of the cells and the formation of the cyprids. The results are compared with those on the developing embryos of the same species.
INTRODUCTION
Changes in the major biochemical components of cirripede eggs during their incubation in the mantle cavity of the adult have so far been studied in several species (e.g. Barnes, 1965; Achituv & Barnes, 1976, 1978b), of which one is the tropical barnacle Tetraclita squamosa rufotincta (Achituv & Barnes, 1978b). The gross biochemical changes during transition from the first nauplius stage to the second has been investigated in the boreo-arctic cirripede Balanus balanoides (Achituv et al., 1980) and Holland & Walker (1975) studied the biochemical composition of the cypris larvae of this same species. Our knowledge of the metabolism in tropical planktonic communities and especially tropical cirripedes is somewhat inadequate. Such information is important for an understanding of the physiological and morphological development of planktonic larvae and their relation to the environmental conditions. Tetraclita squamosa rufotincta occupies the midlittoral zone on the rocky shores of the Red Sea (Safriel & Lipkin, 1964; Achituv, 1972). The breeding season of the population in the Gulf of Elat begins in October and continues through November and December. Each animal probably produces more than one brood during this period (Achituv & Barnes, 1978a). It was found (Achituv & Barnes, 1978b) that at the end of the embryonic development there is relatively more nutrient material left in the embryos of T.s. rufotincta than in any other Balanomorpha so far studied. It was suggested that this may be of survival value to the nauplius stages in the nutrient-poor water of the Gulf of Elat. 241
242
Y.ACHITUV
The possibility of rearing the larvae of T.s. rufotincta Pilsbry through the six nauplius stages to the cypris facilitated the investigation of some aspects of the biochemical changes taking place in the larvae during their development.
MATERIALSANDMETHODS
Animals were collected in Elat and transported to the laboratory. Ripe egg lamellae containing embryos just prior to hatching were removed from the mantle cavity, gently broken down to separate the eggs, washed in sea water and put into a dish containing fresh sea water. The hatching stage I nauplii were concentrated by attraction to light and pipetted into 1-litre beakers of sea water containing 0.005% chloramphenicol. The nauplii were kept at 20 &-1 “C; the water was gently aerated and no food was added to the cultures. To obtain the first sample newly hatched nauplii (after attraction to light) were transferred directly into ice-cold sea water. For subsequent samples viable nauplii were removed after attraction to light, washed in sea water and the exuvia removed by decantation. The nauplii were concentrated by filtration and lightly blotted. A subsample was then weighed, dried at 105 “C for 24 h, and weighed again for the determination of the oven dry weight. Another sample was weighed, preserved, and then transferred to a Bogorov tray, and the nauplii counted. This enables the number of nauplii per unit weight to be calculated. The same subsample was retained for the determination of the stage of development of the nauplii. The remainder was freeze-dried and used for the biochemical analyses. Protein was extracted as described by Achituv & Barnes (1976) and estimated by the method of Lowry et al. (1951). Carbohydrate was estimated by the method of Kemp & Kit van Heijningen (1954). The sulphophosphovanillin method of Barnes & Blackstock (1973) was used for the estimation of total lipids. DNA and RNA were extracted as described by Volkin & Cohn (1954) and their method was used for the estimation of RNA. Burton’s method (1956) was used for DNA estimation.
RESULTSAND
DISCUSSION
The transition from Stage I to Stage II is very rapid and occurs almost immediately after the embryos are liberated as free swimming larvae. This transition is so rapid that even when nauplii are collected immediately after hatching into ice-cold sea water, 85-90x were already at Stage II. Although hatching was more or less synchronized and occurred within a few hours after removal of the ripe egg lamellae from the adult animal, the rate of development was variable; usually a mixture of two successive nauplius stages were found in the same culture, with one of the stages predominating. The results are, therefore, presented by age, rather than by stage. The composition of the samples and their age are shown in Table I.
BIOCHEMICAL
Oven
dry weight
It is evident hatching.
that
During
COMPOSITION
OF TETRACLITA
expressed
in mg/106 nauplii
of nauplii
there
is a weight
decrease
are given
of 14.6% within
the next 4 days there is apparently
243
NAUPLII
no change.
in Table
I.
the first 24 h after The transitions
to
stage VI and then to the cypris are again associated with weight loss. The moisture content of the nauplii increases from 51 to 65% in the last nauplius stage. This is probably associated with the increase in size during development of the nauplii (Barnes & Achituv, in prep.), which is not associated with a gain in dry weight. TABLE
Terraclita squamosa rufotincta: Age of culture and nauplius stage Just hatched, 1 day, Stages 3 days, Stages 5 days, Stages 7 days, Stage Cyprid
Stages I + II II + III III + IV IV + V VI
I
cmen dry weight and water content Oven dry wt. (mg/l O6 nauplii) 12 310 10515 10 626 10693 9 706 8 232
of cultured
Change
nauplii. Water content
(1”)
(5”)
- 14.6 - 13.7 - 13.2 -21.2 - 33.1
51 56 55 65 65 66
The amounts of the major biochemical entities at the various ages, expressed in terms of mg/106 nauplii, are given in Table II. Protein is not utilized very much during the first 5 days of larval development, but there is a marked decrease towards the transition to the cypris stage. Carbohydrate values are very low and almost at the lower limit of the method of estimation; with development there is a slight increase in carbohydrate content. Barnes et al. (1963) showed that protein can provide the glucose and nitrogen needed for the formation of chitin and so the decrease in protein (and related increase in carbohydrate) is most probably associated not only with energy expenditure, but also with the formation of chitin by the cyprids. After the initial loss of lipids there is practically no loss during the development
of the nauplii.
The apparent
slight increase
in the last stage must be
due to experimental error. Calculating the energy supplied by the protein and lipids used (Table III) it is evident that protein contributes most of the energy (almost 80% of the total energy used by the nauplii) for maintenance and development. That contributed by carbohydrate can be neglected. The use of protein as the major component followed by lipid is in agreement with the results of Achituv et al. (1980) for Balanus balanoides. Lipid values remain high up to the cypris stage. Holland & Walker (1975) showed that non-feeding free-swimming cyprids of B. balanoides use their lipid reserves as an energy source for maintenance, particularly when prevented from settling. The fact that the lipid content of Tetraclita squamosa cyprids is still high may be because the sample for analysis was taken before the cyprid had expended any appreciable amount of energy in swimming.
5724
5714 568 1 5581 5091 4873 328 1
1 3 5 7 8 Cyprid
mg/106 nauplii
0
(days)
Age of culture
Protein
0.2 -0.8 -2.5 -11.1 - 14.9 -42.7
_
%
Cumul. change
2832 2842 2621 2592 2665 2790
3165
mg/106 nauplii
Lipids
_
-10.5 - 10.2 -17.2 -18.1 - 15.8 -11.8
_
%
Cumul. change
Tetraclita squamosa rufotincta: biochemical
7.2 9.2 11.7 10.4 15.9 24.0 39.0
nauplii;
+ 27.8 +62.5 + 44.4 +120 +233 +441
%
Cumul. change
Carbohydrate
of cultured
mg/ 1O6 nauplii
composition
TABLE II
30 34.5 45 105 109 109 109
mg/106 nauplii
DNA
+15 +50 + 250 +263 + 263 +263
%
Cumul. change
stages are as defined
+ 60.6 +61.2
_ 464 466
%
Cumul. change
+ 12.3 + 16.6 + 24.2
RNA
289 326 337 356
mg/l@ nauplii
in Table I.
2
6 G
re
BIOCHEMICAL
COMPOSITION
OF TETRACLZTA
NAUPLII
245
Although no food was added to the cultures during development of the larvae, the conditions were not aseptic and there is a possibility that protozoans and bacteria introduced by the hatching nauplii served as a source of food. The contribution of these organisms is likely to be small, as water in the culture media was changed frequently. Moreover, the larvae decrease in weight during development, while samples of Balanus balanoides and B. crenatus, known to have fed gain weight, and all other biochemical constituents (Barnes & Achituv, unpubl. data). This confirms the suggestion (Achituv & Barnes, 1978b) that in the nutrient-poor water of the Gulf of Elat Tetraclita squamosa nauplii rely on their reserves rather than on exogenous food supplies. TABLE III Terraclita squamosa rufotincra: changes in energy content (kcaljlo6 nauplii) of protein and lipids in different ages of nauplii cultures; conversion factors used, 5.65 kcaI/g protein and 9.45 kcal/g lipids. Cumulative change
Age (days)
Protein (kcal/106 nauplii)
Lipids (kcal/ 1O6 nauplii)
Total
3 5 7 8 Cyprid Total kcal used
32.34 32.28 32.10 31.53 28.76 27.53 18.54 13.80
29.91 26.76 26.86 24.77 24.49 25.18 26.37 3.54
62.25 59.04 58.96 56.30 53.25 52.71 44.91 17.34
0
1
(%)
-
3.21 3.29 5.95 9.00 9.54 - 17.34
There was an increase in DNA values during the first five to seven days of nauplius development, which suggests that beyond this stage most of the development is concerned mainly with the organization of the cells and the formation of the cyprid. Morphological observations (Barnes & Achituv, in prep.) confirm this suggestion, because at this stage the cypris limbs begin to be visible inside the thin cuticle of the nauplius. RNA values rise during development, especially in the later stages. According to Holland & Hannant (1973) in cirripede larvae RNA is concerned with high growth rate although this is not invariably the case with, for instance, oyster larvae. As formerly shown, at this stage of development, rate of protein utilization is the highest, probably associated with chitin formation of the cyprids. The RNA/DNA ratios, particularly for the later samples (5 days, 3.39; 8 days, 4.28), compare favourably with those of 3.4, 3.3, and 2.9 quoted by Holland & Hannant (1973) for Elminius modestus, Balanus balanoides, and B. hameri, respectively. The results of the analyses reported above are in good agreement with those given by Achituv & Barnes (1978b) for the developing embryos of Tetraclita squamosa rufotincta from Elat. It was found that at the end of the embryonic
246
Y.ACHITUV
development there were 62.9 kcal/l@ eggs, which compares well with the figure for Stage I nauplii of 62.26 (Table III). It seems that the speculation of the large food reserves being necessary for the survival of the nauplii during development in the nutrient-poor water of the Gulf of Elat was justified.
ACKNOWLEDGEMENT
The author wishes to thank Miss A. Wortzlavski for technical assistance.
REFERENCES ACHITUV, Y., 1972. The zonation of Tetrachihamalus ohlirteratus Newman and Tetraclita squamosa rufotincta Pilsbry in the Gulf of Elat, Red Sea. J. e.up. mar. Biol. Ecol., Vol. 8, pp. 73-81. ACHITUV, Y. & H. BARNES, 1978b. Studies in the biochemistry of cirripede eggs. VI. Changes in the general biochemical composition during development of Chthamalus stellatus (Poli). J. e’_vp. mar. Biol. Ecol., Vol.22, pp. 263-267. ACHITUV, Y. & H. BARNES, 1978a. Some observations on Tetraclita squamosa ruforincra Pilsbry. J. exp. mar. Biol. Ecol., Vol.31, pp. 315-324. ACHI~UV, Y. & H. BARNES, 1978b. Studies in the biochemistry of cirripede eggs. VI. Changes in the general biochemical composition during development of Tetracta squamosa rufotincta Pilsbry, Balanus perforatus Burg., and Pollicipes cornucopia Darwin. J. exp. mar. Biol. Ecol., Vol. 32, pp. 171-176. ACHI-~UV, Y., J. BLACKSTOCK, M. BARNES & the late H. BARNES, 1980. Some biochemical constituents of stage I and II nauplii of Balanus balanoides (L.) and the effect of anoxia on stage I. J. exp. mar. Biol. Ecol., Vol.42, pp. 1-12. BARNES, H., 1965. Studies in the biochemistry of cirripede eggs. I. Changes in the general biochemical composition during development of Balanus balanoides and B. balanus. J. mar. biol. Ass. U.K., Vol. 45, pp. 321-339. BARNES, H., M. BARNES & D. M. FINLAYSON, 1963. The seasonal changes in body weight, biochemical composition, and oxygen uptake of two common boreo-arctic cirripedes, Balanus balanoides (L.) and B. balanus (L.) da Costa. J. mar. biol. Ass. U.K., Vol.43, pp. 185-211, BARNES, H. &J. BLACKSTOCK, 1973. Estimation of lipids in marine animal tissues: detailed investigation of the sulphophosphovanillin method for total lipids. J. exp. mar. Biol. Ecol., Vol.12, pp. 103-l 18. BURTON, K., 1956. A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J., Vol. 62, pp. 315-323. HOLLAND, D. L. & P. J. HANNANT, 1973. Addendum to a micro-analytical scheme for the biochemical analysis of marine invertebrate larvae. J. mar. biol. Ass. U.K., Vol. 53, pp. 833-838. HOLLAND, D. L. & G. WALKER, 1975. The biochemical composition of the cypris larva of the barnacle Balanus balanoides L.J. Cons. perm. int. Explor. Mer, Vol. 32, pp. 162-165. KEMP, A. & A. J. M. KIT VAN HEIJNIGEN, 1954. A calorimetric micro-method for the determination of glycogen in tissues. Biochem. J., Vol.56, pp. 646-648. LOWRY, 0. H., N. J. ROSENBOROUCJH, A. L. FARR & R. J. RANDALL, 1951. Protein measurement with the Folin phenol reagent. J. biol. Chem., Vol.193, pp. 265-275. SAFRIEL, U. & Y. LIPKIN, 1964. Note on the intertidal zonation of the rocky shores of Eilat (Red Sea, Israel). Isr. J. Zool., Vol. 13, pp. 187-190. VOLKIN, E. & W.E. COHN, 1954. Estimation of nucleic acids. In, Methods of biochemical analysis, Vol. I, edited by D. Glick, Interscience Publishers Inc., New York, pp. 287-303.
THE BIOCHEMICAL
pp. 241-246 Press
COMPOSITION
OF TETRACLITA
Y. Department
OF THE NAUPLIUS
SQUAMOSA
of Life Sciences,
RUFOTINCTA
STAGES
Pilsbry
ACHITUV Bar-Ilan
University,
Ramat-Gan,
Israel
Abstract: The biochemical composition of laboratory-reared nauplius stages of Tetraclita squamosa rufotincta Pilsbry during development is described. The larvae lose weight progressively during development. Proteins contribute most of the energy during development and it is suggested that protein serves as the source of nitrogen and glucose for the formation of chitin. Lipids contribute some energy during the initial stages of development, and later may serve as an energy source for the cyprids. The rise in RNA values is related to the increase in protein metabolism, particularly in the later stages. DNA values increase only during the early stages suggesting that later development is concerned mainly with organization of the cells and the formation of the cyprids. The results are compared with those on the developing embryos of the same species.
INTRODUCTION
Changes in the major biochemical components of cirripede eggs during their incubation in the mantle cavity of the adult have so far been studied in several species (e.g. Barnes, 1965; Achituv & Barnes, 1976, 1978b), of which one is the tropical barnacle Tetraclita squamosa rufotincta (Achituv & Barnes, 1978b). The gross biochemical changes during transition from the first nauplius stage to the second has been investigated in the boreo-arctic cirripede Balanus balanoides (Achituv et al., 1980) and Holland & Walker (1975) studied the biochemical composition of the cypris larvae of this same species. Our knowledge of the metabolism in tropical planktonic communities and especially tropical cirripedes is somewhat inadequate. Such information is important for an understanding of the physiological and morphological development of planktonic larvae and their relation to the environmental conditions. Tetraclita squamosa rufotincta occupies the midlittoral zone on the rocky shores of the Red Sea (Safriel & Lipkin, 1964; Achituv, 1972). The breeding season of the population in the Gulf of Elat begins in October and continues through November and December. Each animal probably produces more than one brood during this period (Achituv & Barnes, 1978a). It was found (Achituv & Barnes, 1978b) that at the end of the embryonic development there is relatively more nutrient material left in the embryos of T.s. rufotincta than in any other Balanomorpha so far studied. It was suggested that this may be of survival value to the nauplius stages in the nutrient-poor water of the Gulf of Elat. 241
242
Y.ACHITUV
The possibility of rearing the larvae of T.s. rufotincta Pilsbry through the six nauplius stages to the cypris facilitated the investigation of some aspects of the biochemical changes taking place in the larvae during their development.
MATERIALSANDMETHODS
Animals were collected in Elat and transported to the laboratory. Ripe egg lamellae containing embryos just prior to hatching were removed from the mantle cavity, gently broken down to separate the eggs, washed in sea water and put into a dish containing fresh sea water. The hatching stage I nauplii were concentrated by attraction to light and pipetted into 1-litre beakers of sea water containing 0.005% chloramphenicol. The nauplii were kept at 20 &-1 “C; the water was gently aerated and no food was added to the cultures. To obtain the first sample newly hatched nauplii (after attraction to light) were transferred directly into ice-cold sea water. For subsequent samples viable nauplii were removed after attraction to light, washed in sea water and the exuvia removed by decantation. The nauplii were concentrated by filtration and lightly blotted. A subsample was then weighed, dried at 105 “C for 24 h, and weighed again for the determination of the oven dry weight. Another sample was weighed, preserved, and then transferred to a Bogorov tray, and the nauplii counted. This enables the number of nauplii per unit weight to be calculated. The same subsample was retained for the determination of the stage of development of the nauplii. The remainder was freeze-dried and used for the biochemical analyses. Protein was extracted as described by Achituv & Barnes (1976) and estimated by the method of Lowry et al. (1951). Carbohydrate was estimated by the method of Kemp & Kit van Heijningen (1954). The sulphophosphovanillin method of Barnes & Blackstock (1973) was used for the estimation of total lipids. DNA and RNA were extracted as described by Volkin & Cohn (1954) and their method was used for the estimation of RNA. Burton’s method (1956) was used for DNA estimation.
RESULTSAND
DISCUSSION
The transition from Stage I to Stage II is very rapid and occurs almost immediately after the embryos are liberated as free swimming larvae. This transition is so rapid that even when nauplii are collected immediately after hatching into ice-cold sea water, 85-90x were already at Stage II. Although hatching was more or less synchronized and occurred within a few hours after removal of the ripe egg lamellae from the adult animal, the rate of development was variable; usually a mixture of two successive nauplius stages were found in the same culture, with one of the stages predominating. The results are, therefore, presented by age, rather than by stage. The composition of the samples and their age are shown in Table I.
BIOCHEMICAL
Oven
dry weight
It is evident hatching.
that
During
COMPOSITION
OF TETRACLITA
expressed
in mg/106 nauplii
of nauplii
there
is a weight
decrease
are given
of 14.6% within
the next 4 days there is apparently
243
NAUPLII
no change.
in Table
I.
the first 24 h after The transitions
to
stage VI and then to the cypris are again associated with weight loss. The moisture content of the nauplii increases from 51 to 65% in the last nauplius stage. This is probably associated with the increase in size during development of the nauplii (Barnes & Achituv, in prep.), which is not associated with a gain in dry weight. TABLE
Terraclita squamosa rufotincta: Age of culture and nauplius stage Just hatched, 1 day, Stages 3 days, Stages 5 days, Stages 7 days, Stage Cyprid
Stages I + II II + III III + IV IV + V VI
I
cmen dry weight and water content Oven dry wt. (mg/l O6 nauplii) 12 310 10515 10 626 10693 9 706 8 232
of cultured
Change
nauplii. Water content
(1”)
(5”)
- 14.6 - 13.7 - 13.2 -21.2 - 33.1
51 56 55 65 65 66
The amounts of the major biochemical entities at the various ages, expressed in terms of mg/106 nauplii, are given in Table II. Protein is not utilized very much during the first 5 days of larval development, but there is a marked decrease towards the transition to the cypris stage. Carbohydrate values are very low and almost at the lower limit of the method of estimation; with development there is a slight increase in carbohydrate content. Barnes et al. (1963) showed that protein can provide the glucose and nitrogen needed for the formation of chitin and so the decrease in protein (and related increase in carbohydrate) is most probably associated not only with energy expenditure, but also with the formation of chitin by the cyprids. After the initial loss of lipids there is practically no loss during the development
of the nauplii.
The apparent
slight increase
in the last stage must be
due to experimental error. Calculating the energy supplied by the protein and lipids used (Table III) it is evident that protein contributes most of the energy (almost 80% of the total energy used by the nauplii) for maintenance and development. That contributed by carbohydrate can be neglected. The use of protein as the major component followed by lipid is in agreement with the results of Achituv et al. (1980) for Balanus balanoides. Lipid values remain high up to the cypris stage. Holland & Walker (1975) showed that non-feeding free-swimming cyprids of B. balanoides use their lipid reserves as an energy source for maintenance, particularly when prevented from settling. The fact that the lipid content of Tetraclita squamosa cyprids is still high may be because the sample for analysis was taken before the cyprid had expended any appreciable amount of energy in swimming.
5724
5714 568 1 5581 5091 4873 328 1
1 3 5 7 8 Cyprid
mg/106 nauplii
0
(days)
Age of culture
Protein
0.2 -0.8 -2.5 -11.1 - 14.9 -42.7
_
%
Cumul. change
2832 2842 2621 2592 2665 2790
3165
mg/106 nauplii
Lipids
_
-10.5 - 10.2 -17.2 -18.1 - 15.8 -11.8
_
%
Cumul. change
Tetraclita squamosa rufotincta: biochemical
7.2 9.2 11.7 10.4 15.9 24.0 39.0
nauplii;
+ 27.8 +62.5 + 44.4 +120 +233 +441
%
Cumul. change
Carbohydrate
of cultured
mg/ 1O6 nauplii
composition
TABLE II
30 34.5 45 105 109 109 109
mg/106 nauplii
DNA
+15 +50 + 250 +263 + 263 +263
%
Cumul. change
stages are as defined
+ 60.6 +61.2
_ 464 466
%
Cumul. change
+ 12.3 + 16.6 + 24.2
RNA
289 326 337 356
mg/l@ nauplii
in Table I.
2
6 G
re
BIOCHEMICAL
COMPOSITION
OF TETRACLZTA
NAUPLII
245
Although no food was added to the cultures during development of the larvae, the conditions were not aseptic and there is a possibility that protozoans and bacteria introduced by the hatching nauplii served as a source of food. The contribution of these organisms is likely to be small, as water in the culture media was changed frequently. Moreover, the larvae decrease in weight during development, while samples of Balanus balanoides and B. crenatus, known to have fed gain weight, and all other biochemical constituents (Barnes & Achituv, unpubl. data). This confirms the suggestion (Achituv & Barnes, 1978b) that in the nutrient-poor water of the Gulf of Elat Tetraclita squamosa nauplii rely on their reserves rather than on exogenous food supplies. TABLE III Terraclita squamosa rufotincra: changes in energy content (kcaljlo6 nauplii) of protein and lipids in different ages of nauplii cultures; conversion factors used, 5.65 kcaI/g protein and 9.45 kcal/g lipids. Cumulative change
Age (days)
Protein (kcal/106 nauplii)
Lipids (kcal/ 1O6 nauplii)
Total
3 5 7 8 Cyprid Total kcal used
32.34 32.28 32.10 31.53 28.76 27.53 18.54 13.80
29.91 26.76 26.86 24.77 24.49 25.18 26.37 3.54
62.25 59.04 58.96 56.30 53.25 52.71 44.91 17.34
0
1
(%)
-
3.21 3.29 5.95 9.00 9.54 - 17.34
There was an increase in DNA values during the first five to seven days of nauplius development, which suggests that beyond this stage most of the development is concerned mainly with the organization of the cells and the formation of the cyprid. Morphological observations (Barnes & Achituv, in prep.) confirm this suggestion, because at this stage the cypris limbs begin to be visible inside the thin cuticle of the nauplius. RNA values rise during development, especially in the later stages. According to Holland & Hannant (1973) in cirripede larvae RNA is concerned with high growth rate although this is not invariably the case with, for instance, oyster larvae. As formerly shown, at this stage of development, rate of protein utilization is the highest, probably associated with chitin formation of the cyprids. The RNA/DNA ratios, particularly for the later samples (5 days, 3.39; 8 days, 4.28), compare favourably with those of 3.4, 3.3, and 2.9 quoted by Holland & Hannant (1973) for Elminius modestus, Balanus balanoides, and B. hameri, respectively. The results of the analyses reported above are in good agreement with those given by Achituv & Barnes (1978b) for the developing embryos of Tetraclita squamosa rufotincta from Elat. It was found that at the end of the embryonic
246
Y.ACHITUV
development there were 62.9 kcal/l@ eggs, which compares well with the figure for Stage I nauplii of 62.26 (Table III). It seems that the speculation of the large food reserves being necessary for the survival of the nauplii during development in the nutrient-poor water of the Gulf of Elat was justified.
ACKNOWLEDGEMENT
The author wishes to thank Miss A. Wortzlavski for technical assistance.
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