Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae

Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae

Aquaculture, 50 (1985) 39-49 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands 39 EFFECTS OF DIEiTARY LIPIDS, FATTY ACIDS, AN...

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Aquaculture, 50 (1985) 39-49 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

39

EFFECTS OF DIEiTARY LIPIDS, FATTY ACIDS, AND PHOSPHOLIPIDS ON GROWTH AND SURVIVAL OF PRAWN (PENAEUS JAPONICUS) LARVAE

AK10 KANAZAWA, SHIN-ICHI TESH~A Faculty

of Fisheries,

and MINESHI SAKA~OTO

University of Kagoshima,

4-50-20

~h~~o~rntu,

Kagoshi~a

890

fJwW (Accepted

2 August 1985)

ABSTRACT Kanazawa, A., Teshima, S. and Sakamoto, M., 1985. Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae. Aquaculfure, 50: 39-49. The effects of lipids, fatty acids, and phospholipids on growth and survival of larvae of the prawn, Penaeus japonicus, were examined using purified diets containing carrageenan as a binder. P. japonicus larvae did not metamorphose to post-larvae and died in 7 days when fed the diets containing no phospholipid. Growth and survival rate of the prawn larvae were improved by the addition of soybean phosphatidylcholine (PC) to the diets containing 18:lw9 and essential fatty acids (EFA), or pollack liver oil (PLO) as lipid sources. These results suggest that f? japonicus larvae probably require dietary sources of some phospholipids for growth and survival. The efficacy of phospholipids in improving growth and survival varied with the kinds and sources of phospholipids. Bonito-egg PC, soybean PC, and soybean phosphatidylinositol (PI) had a high efficacy, whereas chickenegg PC was ineffective. Ovine-brain phosphatidylserine, bonito-egg phosphatidylethanolamine (PE), and ovine-brain PE improved slightly growth and survival of P. japonicus larvae. The PC and PI containing high proportions of w6- and w3-fatty acids as constituents were thought to be effective in improving growth and survival of the prawn larvae. The optimum level of soybean PC for P. japonicus larvae varied with the kinds of coexistent dietary lipids. The best growth and survival were attained on the diets containing 6.0% soybean PC when 18:lw9 and 1.0% highly unsaturated fatty acids were used as basal lipids. But the inclusion of 3.5% soybean PC was enough to attain optimum growth and survival when PLO was used as the lipid source. The present study did not give a clear picture of the EFA requirements of P. juponicws larvae, although they seemed to vary with dietary phospholipid levels.

INTRODUCTION

The lipid nutrition of crustaceans is unique in that some, such as the prawn, Penaeus juponicus, and the American lobster, Homarus americanus, require sterols (Kanazawa et al., 1971; Teshima, 1972; Caste11et al., 1975) and phospholipids (Kanazawa et al., 197913; Conklin et al., 1980; D’Abramo

0044-8486185/$03,30

0 1985 Elsevier Science Publishers B.V.

40

et al., 1981) for normal growth and/or survival. However, the above information has been obtained with juveniles, and little is known of the nutritional requirements of larval crustaceans. Recently, we have succeeded in rearing P. japonicus larvae from zoeae to post-larvae with defined diets containing carrageenan as a binder (carrageenan MBD) (Kanazawa et al., 1982; Teshima and Kanazawa, 1982). In the preceding study, we have shown, by feeding trials using a carrageenan MBD, that cholesterol or other sterols are essential for growth and survival of P. japonicus larvae, suggesting the possible transformation of some dietary CZs and CZ9 sterols to cholesterol in the prawn body (Teshima et al., 1983). It has also been demonstrated that phospholipids are important nutrients for prawn larvae as for fish larvae (Kanazawa et al., 1981, 1983a). The optimum levels of cholesterol and soybean phospholipids for growth and survival of P. japonicus larvae were estimated to be 1.0% and 3.0%, respectively, when pollack liver oil was used as the basal lipid source (Teshima et al., 1982). The present study is designed to clarify the dietary value of various lipids and so enhance our understanding of lipid nutrition in larval stages of crustaceans. MATERIALS

AND METHODS

Three experiments were conducted using the zoeal larvae of P. japonicus. P japonicus nauplii were obtained from five egg-bearing females in July (experiment I), August (experiment II), and September (experiment III), 1981. They were reared with the diatom, Chaetoceros gradis, until zoea, or zoeaz stages, and then divided into lots of 100 larvae into l-liter beakers for feedings trials, The feeding and rearing techniques were similar to those reported previously (Jones et al., 1979; Teshima et al., 1982). The zoeal or zoeaz larvae of P. japonicus were fed the various test diets (Tables 1, 2, 3 and 4) at 26-27°C. All test diets contained carrageenan MBD, and were prepared by the same method as described previously (Teshima and Kanazawa, 1982) TABLE

1

Composition

of the basal ration

Ingredienta

(%)

Ingredient

(%)

Casein Glucose Sucrose &-Starch Glucosamine HCl Sodium nitrate Sodium succinate L-Methionine

50.0 5.5 10.0 4.0 0.8 0.3 0.3 0.5

L-Tryptophane Cholesterol Minerals Vitamins Lipids Cellulose

0.5 0.5 8.0 3.2 8.0-16.5 Equal to 100

+ Carrageenan

5.0

aThe basal composition P. japonicus (Kanazawa

of ingredients et al., 1977).

was similar to that of the purified

diet for juvenile

(particle sizes: 60 pm for zoeae, 60-125 pm for mysis). P. japonicus larvae were given the test diets at the concentration of 0.08 mg/larva twice per day (Hirata et al., 1975). The control group was provided C. gracilis (5-7 X 10J cells/ml of water) during zoeal stages and Arternia salina nauplii (500 nauplii/larvae) during mysis stages. During the feeding period, the survival rate of P. japonicus larvae, and the larval stage (Hudinaga, 1942) of 10 random samples were determined every day. In this paper, the larval stages were classified as the following growth indexes: 1, zoeal; 2, zoea2; 3, zoea3; 4, mysis,; 5, mysisz; 6, mysis3; 7, postlarva,. All living larvae were counted and staged at the end of feeding trials when one of the experimental groups reached post-larvai. Statistical analysis of the data on growth indexes and survival rates was by a Chi-square test for experiments I and II and by an analysis of variance for experiment III (according to Steel and Torrie, 1960). The fatty acid composition of dietary lipids was determined by gas-liquid chromatography on 10% DEGS and 5.0% Shinchrom E-71 as mentioned previously (Teshima et al., 1976). RESULTS

Dietary value of lipids and fatty acids Experiment I was conducted to examine the dietary value of various lipids. The zoeaz larvae were reared on the test diets (Table 2). Pollack liver oil (PLO) (diet 3) and soybean oil (diet 4) sustained growth and survival of P. japonicus larvae effectively more than oleic acid (18:lw 9) (diet 2). A superior dietary value of PLO and soybean oil may be due to the inclusion of EFA such as linoleic (18:2w6), linolenic (18:3w3), or w3-highly unsaturated fatty acids (HUFA) in these lipids as pointed out in P. japonicus juveniles (Kanazawa et al., 1977). However, the addition of 1.0% levels of EFA such as 18:2w6, 18:303, HUFA, 18:2w6-18:3w3 (l:l), or 18:2w6 -HUFA (1:l) to the diets confining 7.0% 18:1o9 as the lipid source (diets 6 to 10) did not improve growth and survival of the larvae. All larvae died in 7 days without metamorphosis to post-larva1 when fed the diets containing no phospholipid (diets 2 to 10). Growth and survival of the prawn larvae were improved by a supplement of 1.0% soybean phosphatidylcholine (PC) to the diets containing 6.0% 18:lw9 and 1.0% of either 18:2w6 (diet ll), 18:3w3 (diet 12), or HUFA (diet 13). The prawn larvae had slightly better growth and survival with diets 12 and 13 than with diet 11. This may indicate that w3-fatty acids are effective for P. japonicus larvae as EFA rather than w6-fatty acids as demonstrated in the juveniles (Kanazawa et al., 1979a). However, the EFA requirements of P. japonicus larvae should be estimated more reliably in connection with the types and concentrations of dietary phospholipids in future. Growth index and survival rate of the prawn larvae were significantly (P <0.05) improved by increasing the soybean PC levels from 1.0% to 6.0% in

42 TABLE Effects Diet no.

2 of dietary

Dietary

and survival of P.japonicus larvae

lipids on growth

Growth index

lipid

Survival (%)

Observation day*

No food (starved) 8.0% 18:lw9 8.0% PLO (pollack liver oil) 8.0% soybean oil 4.0% PLO + 4.0% soybean oil

2.0 3.2 4.5 5.0 5.5

0 0

7 3 5

2 4 6 6 6

2 6 I 8 9 10

8.0% 7.0% 7.0% 7.0% 7.0% 7.0%

18:lw9 18:lw9 18:lw9 18:lw9 18:lw9 18:lw9

+ + + + +

1.0% 1.0% 1.0% 0.5% 0.5%

18:2w6 18:3w3 HUFAb 18:2w6 18:2w6

+ 0.5% 18:3w3 + 0.5% HUFA

3.2 3.7 3.7 4.5 3.5 5.2

0 0 0 3 0 4

4 4 5 6 5 6

11 12 13 14 15 16

6.0% 6.0% 6.0% 6.0% 6.0% 6.0%

18:lw9 18:lw9 18:lw9 18:lw9 18:lw9 18:lw9

+ + + + + +

1.0% 1.0% 1.0% 1.0% 1.0% 1.0%

18:2w6 18:3w3 HUFA HUFA HUFA HUFA

+ 1.0% Soybean + 1.0% Soybean + 1.0% Soybean + 3.0% Soybean + 6.0% Soybean + 9.0% Soybean

4.2 4.6 5.5 6.7 6.2 6.2

29 46 38 70 87 78

6 6 6 6 6 6

3 17 18 19 20

8.0% PLO 8.0% PLO + 8.0% PLO + 8.0% PLO + Control (live

4.5 6.8 7.0 7.0 7.0

7 61 86 80 83

6 6 6 6 6

1.0% Soybean 3.5% Soybean 7.5% Soybean food)

PC PC PC

PCc PC PC PC PC PC

aZoea, larvae were reared with the test diets until one of experiment groups reached postlarvae,. bHUFA (w3-highly unsaturated fatty acids) was composed of 20:5w3 (49.1%), 22:6w3 (31.5%), 20:4w6 (5.8%), and small amounts of other fatty acids. CSoybean PC (phosphotidylcholine from soybean) was a mixture of phosphatidylcholine (23.6%), phosphatidylethanolamine (30.4%), phosphatidylinositol (18.1%), and other polar lipids.

the diets containing 18:lw9 and 1.0% HUFA (diets 13 to 15). The zoea2 larvae grew up to post-larva, with 80% survival in 7 days when fed diets 14, 15, and 16. A supplement of l.O%, 3.5%, or 7.5% soybean PC to the diet containing 8.0% PLO again resulted in improved growth and survival. The optimum soybean PC level for P. juponicus larvae was suggested to be near 3.5% in diets when PLO was used as the basal lipid. The zoea2 larvae fed diet 18, containing 8.0% PLO and 3.5% soybean PC, had good growth and survival, comparable to those fed the live feeds (control group: diet 20): they grew up to post-larva, with 86% survival in 6 days (Fig. 1).

43

123456 Feeding period (days)

0

*.



123456



Feeding period (days)

Fig. 1. Growth and survival of P. japonicus larvae fed diets containing several levels of soybean PC. Numerals indicate the diet no. (see Table 2). The diets contained the following lipids: 3, 8.0% PLO; 17, 8.0% PLO + 1.0% soybean PC; 18, 8.0% PLO + 3.5% soybean PC; 19,8.0% PLO + 7.5% soybean PC; 20, live food (control group).

Efficacy of various phospholipids in improving growth and survival of P. japonicus larvae As mentioned above, the inclusion of some phospholipids is probably indispensable for growth and survival of P. japonicus larvae. However, it is not known why such crustaceans as P. japonicus and H. americanus require ndietary sources of phospholipids. We assume that: (1) the prawn larvae may have a limited ability for phospholipid biosynthesis from fatty acids and/or diglycerides, (2) dietary phospholipids may take part in the emulsification of dietary lipids such as triglycerides and cholesterol, and/or (3) some types of dietary phospholipids are necessary as constituents of lipoproteins which play an important role in the transport of lipids. In an attempt to understand the metabolic role of dietary phospholipids in larval crustaceans, the efficacy of various phospholipids in improving growth and survival of P.

44 japanicus

larvae was examined in experiment II. The zoeaz larvae were reared on test diets containing 8.0% PLO and 1.0% levels of eight phospholipids from various sources, cytidine-5’-diphosphate choline sodium salt (CDP-choline), or taurocholic acid. The results are shown in Table 3. High values of growth index and survival rate were obtained with the diets supplemented with bonito-egg PC (diet 22), soybean PC (diet 23), and soybean phosphatidylinositol (PI) (diet 28), whereas, chicken-egg PC (diet 21) was ineffective in improving growth and survival of P. japonicus larvae. Ovine-brain phosphatidyl-L-serine (diet 27), bonito-egg phosphatidylethanolamine (PE) (diet 25), and ovine-brain PE (diet 24) slightly improved growth and/or survival of the prawn larvae, but bovine-brain sphingomyelin (diet 26) had no effect.

TABLE Effects

3 of various phospholipids

Diet no.

Dietary

21 22 23 24 25 26 27 28 29 30 3

7.0% PLO 7 .O% PLO 7.0% PLO 7 .O% PLO 7.0% PLO 7.0% PLO 7 .O% PLO 7.0% PLO 7.0% PLO 7.0% PLO 8.0% PLO

on growth

lipida

+ + + + + + + + + +

1.0% chicken-egg PC 1 .O% bonito-egg PC 1.0% soybean PC 1 .O% ovine-brain PE 1.0% bonito-egg PE 1.0% bovine-brain SM 1 .O% ovine-brain PS 1.0% soybean PI 1 .O% CDP-choline 1.0% taurocholic acid

and survival of P.japonicus

larvae

Growth index

Survival (%)

Observation day

4.5 6.4 6.0 5.3 4.7 4.0 5.7 7.0 4.6 4.0 4.5

0 91 72 34 48 0 55 88 0 0 7

6 6 6 6 6 6 6 6 6 6 6

aPLO (pollack liver oil; Riken Vitamin Co.), chicken-egg PC (phosphatidylcholine from chicken-egg yolk; Nakarai Chemicals Co.), bonito-egg PC (phosphatidylcholine from bonito eggs; isolated in this laboratory), soybean PC (phosphatidylcholine from soybean; Nakarai Chemicals Co.), ovine-brain PE (phosphatidylethanolamine from ovine brain; Sigma Chemicals Co.), bonito-egg PE; phosphatidylethanolamine from bonito eggs; isolated in this laboratory), bovine-brain SM (sphingomyelin from bovine brain; Sigma Chemical Co.), ovine-brain PS (phosphatidyl-Lserine from ovine brain; Nakarai Chemicals Co.), soybean PI (phosphatidylinositol from soybean; Sigma Chemical Co.), CDPcholine (cytidine-5’-diphosphate choline sodium salt; Nakarai Chemicals Co.), taurocholic acid (Nakarai Chemicals Co.).

Effects

of soybean

PC and HUFA

levels in diets

In experiment III, feeding trials were carried out in order to examine the effects of soybean PC and HUFA levels on growth and survival of P. japonicus larvae. The zoea, larvae were reared on 12 diets containing varying levels of soybean PC and HUFA (Table 4). The results are given in Table 4

45

TABLE

4

Test diets used in experiment Diet no.

31 32 33 34 35 36 37 38 39 40 41 42

Dietary

III

lipid (%)

w 3-HUFA

Soybean

0 0 0 0.5 0.5 0.5 1.0 1.0 1.0 2.0 2.0 2.0

1.0 3.0 6.0 1.0 3.0 6.0 1.0 3.0 6.0 1.0 3.0 6.0

PC

Survival rate (%)

Growth index

5 37 76 9 57 76 12 74 86 13 20 64

3.2 6.0 6.5 3.5 5.9 7.0 4.0 6.0 7.0 4.0 4.7 6.0

18:lw9 7.0 5.0 2.0 6.5 4.5 1.5 6.0 4.0 1.0 5.0 3.0 0

and Figs. 2 and 3. Although it is impossible to evaluate an interaction between dietary soybean PC and HUFA levels statistically, Figs. 2 and 3 suggest an interaction between the two factors. The survival rates of P. jq&zicus larvae increased with increasing levels of soybean PC in diets except the diet containing 2% HUFA and 3% soybean PC (diet no. 41). The

1 PC

, i II. diets

Fig. 2. Effects of soybean larvae.

japonicus

6

3 Soybean

PC and w 3-HUFA

0

I.0

0 5 wr*

.I'

'I, 1,111

levels in the diets on survival rates (W) of P.

46

effect of dietary HUFA levels on survival rates varied with dietary soybean PC levels. The survival rates of the larvae were not improved with increasing HUFA levels when diets contained 1% or 6% soybean PC, whereas, survival rates did increase with increasing HUFA levels from 0% to 1.0% and decreased at 2.0% HUFA level when diets contained 3.0% soybean PC. The addition of 2.0% HUFA to diets containing 3% and 6% soybean PC reduced the survival rates for some unknown reasons (an overdose of essential fatty acids or contamination by oxidation products of HUFA?). Regarding the growth index (Fig. 3), the dietary soybean PC and HUFA levels had similar effects as observed on the survival rates. The growth indexes were increased with increasing levels of soybean PC in the diets.

:I:-:\. \

hi.

l

i t I

2’ I

3 irl”bc*n

PI

h p

IS1

,l

-,

PC and w3-HUFA

?I.

1 ,” W,A

ldlC+i

Fig. 3. Effects of soybean japonicus larvae.

1 i;

:

)

II,

xl,?t,

levels in the diets on growth index of P.

DISCUSSION The results of experiment I showed that the inclusion of soybean PC in diets improved growth and survival of P. juponicus larvae. Therefore, the larvae probably require dietary sources of some phospholipids for normal growth and survival. Conklin et al. (1980) and D’Abramo et al. (1981) have shown that the inclusion of phospholipids in diets is necessary for the survival of 2% americanus juveniles. D’Abramo et al. (1981) demonstrated that, for H. americanus, none of ovine PE, soy PI, and non-phospholipids such as the hydrolysis products of soy PC, and fatty acids, and taurocholic acid effectively substituted for the refined soy PC in reducing mortality. Thus, the effects of phospholipids on growth and survival of crustaceans seem to vary with the fatty acids present, in addition to the kinds of compounds esterified at the C-3 position with phosphoric acid. As shown in Table 5, soybean PC and bonito-egg PC con-

47

TABLE 5 Fatty acid composition Fatty acida 12:o 14:o 16:0 16:l 18:O 18:lw9 18:2w6 18:3w3 20: lw9 20:4w6 20:5w3 22:4w3 22:5w3 22:6w3 24:l Unidentific :d

Chickenegg PC

(C) of phospholipids used Bonitoegg PC

Soybean PC

_Ib”

1

1

26.7 5.4 7.8 27.2 4.3 0.4 0.6 2.1 1.9 1.3 2.3 -

22.5 1.8 14.6 5.5 0.2 0.6

20.2 3.6 8.8 60.1 1.3

5.3 3.1 1.7 1.0 32.9 -

OVillebrain PE

2.0 20.0 0.6 23.9 26.8 0.2 0.8 1.8 2.3 1.5 2.4

-

a Determined by gas-liquid chromatography 1976). b (-_) Indicates less than 0.2%.

-

8.3

Bonitoegg PE 0.2 25.5 2.2 16.1 6.9 0.3 1.0 0.5 6.0 2.9 1.6 1.3 28.6

Bovinebrain SM

Ovinebrain PS

Soybean PI

2.2 34.3 0.5

2.2

14.0

46.3 32.9

3.4 9.8 64.9 7.3 -

-

2.7 3.3

40.6 7.3

-

0.6 1.1 1.4

-

7.5

on 5.0% Shinchrom E-71 or 10% DEGS (Teshima et al.,

tamed higher proportion of EFA such as 18:2w6, 18:3w 3, or 22:6w3 than chicken-egg PC. Soybean PI also contained large amounts of 18: 2w 6. Therefore, the PC and PI rich in w6- or w3-fatty acids are likely to be effective in improving growth and survival of P. juponicus larvae. Although bonito-egg PE contained large amounts of 22:6w3, the efficacy of this PE was inferior to that of bonito-egg PC. Also, the low proportions of w6- and w 3-fatty acids and the kind of bases at C-3 may be related to the low efficacy of bovine-brain SM and ovine-brain PS. Mammals synthesize phospholipids from 1,2-diglycerides with the aid of CDP-choline (Imai and Sakagami, 1966). P. japonicus larvae, however, were postulated to have a limited ability for biosynthesis of phospholipid because of the inefficacy of CDP-choline in improving growth and survival. A supplement of ursodeoxycholic acid has been reported to result in increased weight gain of P. juponicus juveniles (Deshimaru, 1981). The addition of taurocholic acid improved neither growth nor survival of P. japonicus larvae, as pointed out in H. americanus juveniles (D’Abramo et al., 1981). This may indicate that dietary phospholipids such as PC and PI possibly improve growth and survival of the prawn larvae by effects other than the enhancement of dietary lipid emulsification in the digestive tracts. Previously, we have shown that the hemolymph of P. juponicus juveniles contained highdensity lipoproteins rich in phospholipids as the major lipoproteins (Teshima and Kanazawa, 1980a);and also that the high-density lipoproteins took part in the transport of lipids through the hemolymph (Teshima and Kanazawa, 1980b). Considering the information available, we suspect that the PC and PI containing high levels of w6- and w3-fatty acids possibly serve as the lipid moieties of highdensity lipoproteins in P. japonicus larvae.

48

In the present study both growth and survival of P. japonicus larvae were found to be affected by the dietary soybean PC levels. However, marked effects of supplemental HUFA on survival rates and growth indexes were observed only on diets with 3.0% soybean PC. In experiment III, the best growth indexes and survival rates were attained on the diet containing 6.0% soybean PC, 1.0% HUFA, and 1.0% 18:109 (Figs. 2 and 3); the zoea, larvae grew up to post-larvae, with 87% survival in 8 days. This indicates that the prawn larvae require 6.0% or more soybean PC in the diets for optimum growth and survival. In experiment I, however, the best growth and survival were attained with diets containing 3.5% soybean PC and 8.0% PLO as lipid sources (Table 2). Considering these results, the optimum levels of soybean PC for P. juponicus larvae should be evaluated in relation to the quality of basal lipids. Since some phospholipids used in this study and by D’Abramo et al. (1981) had a low purity, the requirements of phospholipids for crustaceans should be clarified more rigorously by using pure phospholipids of known molecular species. REFERENCES requirements of juvenile Caste& J.D., Mason, EC. and Covey, J.F., 1975. Cholesterol American lobster (Homarus americanus). J. Fish. Res. Board Can., 38: 1431-1435. Conklin, D.E., D’Abramo, L.R., Bordner, C.E. and Baum, N.A., 1980. A successful purified diet for the culture of juvenile lobsters: the effect of lecithin. Aquaculture, 21: 243-250. D’Abramo, L.R., Bordner, C.E., Conklin, D.E. and Baum, N.A., 1981. Essentially of dietary phosphatidylcholine for the survival of juvenile lobsters. J. Nutr., 111: 425431. Deshimaru, O., 1981. Studies on nutrition and diet for prawn, Penaeus japonicus. Mem. Kagoshima Pref. Fish. Exp. Stn., 12: l-118. Hirata, H., Mori, Y. and Watanabe, M., 1975. Rearing of prawn larvae, Penaeus japonicus, fed soy cake particles and diatoms. Mar. Biol., 29: 9-13. Hudinaga, M., 1942. Reproduction, development and rearing of Penaeus japonicus Bate. Jpn. J. Zool., 10: 305-393. Imai, Y. and Sakagami, T., 1966. Metabolism of compound lipids. In: Biochemistry of Lipids, Asakura-shoten, Tokyo, pp. 156-183 (in Japanese). Jones, D.A., Kanazawa, A. and Abdel Rahman, S., 1979. Studies on the presentation of artificial diets for rearing the larvae of Penaeus japonicus Bate. Aquaculture, 17: 3343. Kanazawa, A., Tanaka, N., Teshima, S. and Kashiwada, K., 1971. Nutritional requirements of prawn. II. Requirement for sterols. Bull. Jpn. Sot. Sci. Fish., 37: 211-215. Kanazawa, A., Teshima, S. and Tokiwa, S., 1977. Nutritional requirements of prawn. VII. Effect of dietary lipids on growth. Bull. Jpn. Sot. Sci. Fish., 43: 849-856. Kanazawa, A., Teshima, S., Tokiwa, S. and Ceccaldi, H.J., 1979a. Effects of dietary linoleic and linolenic acids on growth of prawn. Oceanol. Acta, 2: 43-47. Kanazawa, A., Teshima, S., Tokiwa, S., Endo, M. and Abdel Rahman, S., 1979b. Effects of short-necked clam phospholipids on the growth of prawn. Bull. Jpn. Sot. Sci. Fish., 45: 961-965. Kanazawa, A., Teshima, S., Inamori, S., Iwashita, T. and Nagao, A., 1981. Effects of phospholipids on growth, survival rate, and incidence of malformation in the larval ayu. Mem. Fat. Fish., Kagoshima Univ., 30: 301-309.

49 Kanazawa, A., Teshima, S., Sasada, H. and Abdel Rahman, S., 1982. Culture of the prawn larvae with micro-particulate diets. Bull. Jpn. Sot. Sci. Fish., 48: 195-199. Kanazawa, A., Teshima, S., Inamori, S. and Matsubara, H., 1983a. Effects of dietary phospholipids on growth of the larval red sea bream and knife jaw. Mem. Fat. Fish., Kagoshima Univ., 32: 109-114. Kanazawa, A., Teshima, S., Kobayashi, T., Takae, M., Iwashita, T. and Uehara, R., 1983b. Necessity of dietary phospholipids for growth of the larval ayu. Mem. Fat. Fish., Kagoshima Univ., 32: 115-120. Steel, R.G.D. and Torrie, J.H., 1960. Principles and Procedures of Statistics. McGrawHill, New York, pp. 438-375. Teshima, S., 1972. Sterol metabolism in marine crustaceans. Mem. Fat. Fish., Kagoshima Univ., 21: 69-147. Teshima, S. and Kanazawa, A., 1980a. Lipid constituents of serum lipoproteins in the prawn. Bull. Jpn. Sot. Sci. Fish., 46: 57-62. Teshima, S. and Kanazawa, A., 1980b. Transport of dietary lipids and role of serum lipoproteins in the prawn. Bull. Jpn. Sot. Sci. Fish., 46: 51-55. Teshima, S. and Kanazawa, A., 1982. Microparticulate diets for the larvae of aquatic animals. Min. Rev. Data File Fish Res. Kagoshima Univ., 2: 11-22. Teshima, S., Kanazawa, A. and Okamoto, H., 1976. Analysis of fatty acids of some crustaceans. Mem. Fat. Fish., Kagoshima Univ., 25: 41-46. Teshima, S., Kanazavva, A., Sasada, H. and Kawasaki, M., 1982. Requirements of the larval prawn, Penaeus japonicus, for cholesterol and soybean phospholipids. Mem. Fat. Fish., Kagoshima Univ., 31: 193-199. Teshima, S., Kanazawa, A. and Sasada, H., 1983. Nutritional value of dietary cholesterol and other sterols to larval prawns, Penaeus japonicus. Aquaculture, 31: 159-167.