Biochemical compositions and digestive enzyme activities during the embryonic development of prawn, Macrobrachium rosenbergii

Aquaculture 253 (2006) 573 – 582 www.elsevier.com/locate/aqua-online

Biochemical compositions and digestive enzyme activities during the embryonic development of prawn, Macrobrachium rosenbergii Yao Jun-jie, Zhao Yun-long ⁎, Wang Qun, Zhou Zhong-liang, Hu Xian-cheng, Duan Xiao-wei, An Chuan-guang School of Life Science, East China Normal University, Shanghai, 200062, China Received 2 May 2005; received in revised form 21 August 2005; accepted 22 August 2005

Abstract Biochemical composition and digestive enzyme activities of eggs during embryonic development were studied in the freshwater prawn, Macrobrachium rosenbergii. Proteins, lipids and carbohydrates were the main components in the embryos of M. rosenbergii. The proteins in yolk were used mainly as the structural substance, whereas the lipids and carbohydrates were used mainly as the energy sources. Protein content generally increased while lipid and carbohydrate contents decreased during the embryonic development. Seventeen amino acids, including eight essential amino acids, were found in every stage of embryonic development. The ratio of the contents of each essential amino acid (EAA) to total essential amino acid (TAA) remained unchanged during the different stages of embryonic development. The proportional content of glutamic acid was the highest among all the amino acids, and leucine content was the highest among the EAAs. The predominant fatty acids, in terms of relative proportion, were C16:0, C18:1n-9, C18:2n-6, C18:0 and C16:1 in each embryonic development stage. The monounsaturates (MUFA) were the preferentially utilized components of the unsaturates (UFA). C18:1n-9c was mainly used as an energy source during embryonic development, whereas C18:3n-3 and ARA mainly acted as the structural substances in embryos. SFA acted as the main energy source during early stages, from fertilized egg to gastrula stage, and MUFA acted as the main energy source from egg nauplius to egg metanauplius stage. HUFA were used mainly as energy sources during late stages. Of the five digestive enzymes assayed, activities of pepsin, trypsin and amylase were relatively high. Activities of pepsin, trypsin, amylase and cellulase increased during both the early and later embryonic stages, but decreased during the middle stages. The activity of lipase decreased after the gastrula stage. The gastrula stage was a special stage of embryonic development where organ anlage came into being. Activities of pepsin, trypsin, amylase and cellulase reached the highest level during the zoea stage. Variations of biochemical compositions and digestive enzyme activities were closely related to events in morphogenesis during the embryonic development of M. rosenbergii. © 2005 Elsevier B.V. All rights reserved. Keywords: Macrobrachium rosenbergii; Embryonic development; Protein; Lipid; Digestive enzyme activity

1. Introduction Females of some decapods carry their eggs under the abdomen until hatching. The eggs are rich in yolk substances that are used as embryonic development ⁎ Corresponding author. Tel.: +86 21 62232153; fax: +86 62233754. E-mail address: [email protected] (Y. Zhao). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.08.011

progresses. Protein, one of the main components of yolk, plays an important role in both morphogenesis and energy supply in embryos (Holland, 1978; Luo et al., 2004). Lipid content is relatively high in decapod eggs, and is one of the main energy sources. During the stages of embryonic development, lipids are not only an energy source, but also the components of biological membranes and pigments of compound eyes. Lipids play an

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important role in embryonic metabolism as they are the most important energy source and provide at least 60% of the total energy expended by the developing crustacean embryo (Wehrtmann and Graeve, 1998). Knowledge about the utilization of yolk that occurs during embryonic development is essential for a complete understanding of nutritional requirements of egg-bearing females and the early larvae of crustaceans. Macrobrachium rosenbergii is one of the economically important prawns and is cultured in most regions of China. However, little is known about embryo's nutritional metabolism of this species. Inquiries have risen about how yolk is utilized in the process of embryonic development. Some studies have examined biochemical metabolism in the embryonic development of crustaceans (Clarke et al., 1990; Wang et al., 1995; Petersen and Anger, 1997; Chen et al., 1998; Wehrtmann and Kattner, 1998; Gimenez and Anger, 2001). Information regarding changes in biochemical composition and digestive enzyme activity during the embryonic development of M. rosenbergii is scarce. The external morphological characteristics and morphogenesis of organ anlage during the embryonic development of M. rosenbergii have been extensively studied (Zhao et al., 1998a,b). Digestive enzyme activities in developing crustacean embryos have also been investigated (Biesiot, 1986; Subramoniam, 1991; Tian et al., 2003). The present study was designed to investigate the biochemical composition and digestive enzyme activity during the embryonic development of M. rosenbergii. The data obtained from the present investigation provide valuable information to increase knowledge of yolk utilization in relation to organogenesis, and should contribute to an understanding of crustacean embryonic development. 2. Materials and methods 2.1. Sampling Adult prawns of M. rosenbergii were collected at the Flourishing Well-Bred Shrimp Field, Badu, Jiangsu Province from March to April 2004. They were obtained from Burma in October, 2003. After mating, females were selected and cultured within a net-box (1.0 × 1.0 × 0.5 m) in a cultured pool at a water temperature of 28 °C. Egg-bearing females were divided into three groups and each group contained 10 females. Every day, females were fed a combination of a formulated diet (3×) and snails (2×). The embryonic development of M. rosenbergii takes about 28 days. Embryonic development stages were identified under a microscope (LEICA DMLB) with a calibrated micro-

meter eyepiece. According to the criteria presented by Zhao et al. (1998a), there are eight stages of embryonic development: I. Fertilized egg stage, II. Cleavage stage, III. Blastula stage, IV. Gastrula stage, V. Egg nauplius stage, VI. Egg metanauplius stage, VII. Protozoea stage, and VIII. Zoea stage. Egg masses representing all eight stages of development were removed and from the 10 females representing each group and pooled, resulting in three separate samples of egg masses for analysis. Some eggs were placed into Bouin's fixative liquid for histological section and the rest were stored in liquid nitrogen for later biochemical analyses. 2.2. Egg volume and water content Egg volume was calculated using the formula: V = 1 / 6(πW2L) (Turner and Lawrence, 1979). Water content was determined by determining the dry weight of egg, and by relating it to the wet weight of the samples. 2.3. Biochemical composition Content of protein was determined according to the Kjeldahl method as modified by Liu et al. (1999). The content of carbohydrate was determined using the DNS colorimetric method; total lipids (TL) were extracted according to Bligh and Dyer (1959); neutral lipids (NL) and phospholipids were extracted in chloroform– methanol solution. 2.4. Digestive enzyme activity Digestive enzyme activity was determined according to the Pan luqing method (Pan and Wang, 1997), The concentration of protein in enzyme solution was determined using the Coomassie Brilliant Blue method (Li et al., 1994). 2.5. Amino acid analyses Egg proteins were hydrolyzed with 6 N hydrochloric acid (containing 0.1% phenol) in a hydrolyzation tube, which was vacuumed and filled with nitrogen, at 110 °C for 22 h. The reaction mixture was diluted with water to a volume of 50 ml, followed by filtration. A 1 ml of filtered liquid was dried up in a vacuum desiccator (40∼50 °C liquid), followed by dilution with 2 ml distilled water. After drying, the sample was dissolved with 1 ml pH 2.2 buffer. An automatic analyzer (Biochrom 20, Amersham Biosciences) was used to determine amino acid content. Amino acids were identified by

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P < 0.05 (Zar, 1996). The software Statistica (versuion 4.5) were also used analyzing correlation coefficient r value between the egg volume and the water content.

comparison of their retention time with those of specific standards (Sigma). 2.6. Fatty acid analysis

3. Results A capillary gas chromatography method was employed to determine the proportional level of fatty acids. A HP6890 (FID detector) and a SPTM-2380 column (30 m × 0.25 mm × 0.20 ìm) were used in the experiment. The separation was carried out with nitrogen as the carrier gas. Temperature of the column was programmed from 140–240 °C at 4 °C/ min, held for 5 min at 140 °C and 10 min at 240 °C, with a detector at 260 °C. A split injector (50 : 1) at 260 °C was used. Fatty acids were identified by comparison of their retention time to those of chromatographic Sigma standards. Peak areas were determined using the Varian software.

3.1. Egg volume and water content A significant increase in egg volume occurred during the embryonic development of M. rosenbergii. Egg volume ranged from 0.072 mm3 in the fertilized egg stage to 0.106 mm3 in the zoea stage. A similar trend was observed for water content, which increased from 52.59% to 74.84% (Fig. 1). A marked correlation existed between the egg volume and the water content (r = 0.995). Egg volume increased slowly from the fertilized egg stage to the protozoea stage, followed by a significant increase in the zoea stage, from 0.087 to 0.106 mm3.

2.7. Histological section 3.2. Contents of protein, lipid and carbohydrate Eggs of each stage were fixed in Bouin's fixative liquid for 24 h, then dehydrated by series of gradient alcohol. After clarity with methyl salicylate and xylene, and paraffin imbedding, a AO-B20 microtome (Spencer lens Co. USA) was used to obtain 6 μm histological sections. Staining was performed with hematoxylin-eosin.

Levels of lipid and carbohydrate decreased significantly while the content of protein increased as development progressed (Table 1). Content of protein in fertilized egg stage was 18.67 ± 0.22 μg/egg and increased gradually to 23.97 ± 0.51 ug/egg in the protozoea stage. In the zoea stage, the protein content decreased to 20.18 ± 0.49 μg/egg. Content of protein per egg increased 1.51 μg during embryonic development. From the fertilized egg stage to zoea stage, lipid content decreased from 13.52 ± 0.27 to 7.77 ± 0.50 μg/ egg, representing an overall decrease of 42.53%. The content of neutral lipids (NL) decreased by 69.22% as the development progressed, from 8.48 ± 0.27μg/egg in

2.8. Statistical analysis Results are presented as means ± s.d. (n = 3). Differences among means were analyzed by one-way analysis of variance (one-way ANOVA), followed when pertinent by a multiple comparison test (Tukey). Differences were reported statistically significant when

90

0.14 Embryo volume Water content

80 70

0.1

60

0.08

50

0.06

40 30

0.04

20 0.02 0

Water content (%,ww)

Embryo volume (mm3)

0.12

10 I

II

III

IV

V

VI

VII VIII

0

Embryonic development stage Fig. 1. Volume and water content of M. rosenbergii eggs during embryonic development. (I. Fertilized egg stage; II. Cleavage stage; III. Blastula stage; IV. Gastrula stage; V. Egg nauplius stage; VI. Egg metanauplius stage; VII. Protozoea stage; VIII. Zoea stage).

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Table 1 Mean amounts of the main biochemical compounds in the eggs (μg/egg) of Macrobrachium rosenbergii during development (X ± SD, n = 3) Embryonic stages

Protein (μg/egg)

Carbohydrate (μg/egg)

Lipid (μg/egg)

Neutral lipids (μg/egg)

Phospholipids (μg/egg)

Dry weight (μg/egg)

Fertilized egg (I) Cleavage (II) Blastula (III) Gastrula (IV) Egg nauplius (V) Egg metanauplius (VI) Protozoea (VII) Zoea (VII)

18.67 ± 0.22b 18.12 ± 0.22ab 17.71 ± 0.32a 18.65 ± 0.24b 21.52 ± 0.37d 20.77 ± 0.48c 23.97 ± 0.51e 20.18 ± 0.49c

3.31 ± 0.32d 1.00 ± 0.23b 1.39 ± 0.11c 1.22 ± 0.06bc 1.34 ± 0.07c 1.19 ± 0.12bc 0.29 ± 0.05a 0.10 ± 0.02a

13.52 ± 0.27e 12.78 ± 0.10d 12.54 ± 0.23cd 12.68 ± 0.24d 12.45 ± 0.50cd 11.95 ± 0.26bc 11.59 ± 0.38b 7.77 ± 0.50a

8.48 ± 0.27g 7.80 ± 0.03f 7.38 ± 0.08e 6.45 ± 0.39d 4.75 ± 0.28bc 4.49 ± 0.14b 5.11 ± 0.11c 2.61 ± 0.22a

3.43 ± 0.11e 3.29 ± 0.07de 3.07 ± 0.10c 2.81 ± 0.04b 2.88 ± 0.11b 3.09 ± 0.16c 3.23 ± 0.10cd 2.18 ± 0.13a

40.70 ± 0.99d 39.67 ± 0.58d 39.03 ± 1.00d 37.34 ± 0.58c 37.10 ± 0.49c 37.22 ± 0.58c 35.09 ± 0.20b 33.00 ± 0.24a

Different superscript letters within vertical rows represent significant differences (P < 0.05).

the fertilized egg stage to 2.61 ± 0.22 μg/egg in the zoea stage. The content of phospholipids decreased slightly. The concentration of carbohydrate in fertilized egg stage was 3.31 ± 0.32 μg/egg, and decreased sharply to 1.00 ± 0.23 μg/egg in the cleavage stage, and continued to decrease in the protozoea and zoea stages (1.19 ± 0.12, 0.29 ± 0.05 μg/egg, respectively). The carbohydrate content decreased 96.98% during embryonic development.

(NEAA), were identified in each stage (Table 2). Tryptophan and taurine cannot be determined accurately because of hydrolyzation by HCl. The amino acid content, 43.91% of dry weight, was the lowest in the fertilized egg stage and reached the highest level, 53.96%, in the zoea stage. Among all the amino acids, the proportional amount of glutamic acid was the highest, and leucine content was the highest among EAAs. The proportional amount of EAAs, such as leucine, lysine, phenylalanine, threonine, methionine and arginine, increased gradually as development progressed. Leucine content increased from 3.95% to 4.72% of dry

3.3. Amino acid content Seventeen amino acids, including nine essential amino acid (EAA) and eight nonessential amino acids

Table 2 The proportional composition of amino acids of eggs of M. rosenbergii during different stages of the embryonic development (%, dry weight) and the results of statistical analysis Embryonic development stages I Leu Lys Val Ile Phe Thr Met Arg His ΣEAA NEAA Glu Asp Gly Ala Ser Cys Tyr Pro ΣTAA

II a

III a

IV ab

V abc

VI abc

VII abc

VIII c

3.95 ± 0.24 3.81 ± 0.24ab 3.14 ± 0.18ab 2.47 ± 0.27a 2.04 ± 0.27 1.91 ± 0.19 1.22 ± 0.18a 3.19 ± 0.12a 1.53 ± 0.26 23.26 ± 1.92a

4.06 ± 0.27 3.60 ± 0.11a 2.97 ± 0.20a 2.33 ± 0.33a 2.03 ± 0.16 2.06 ± 0.28 1.73 ± 0.16d 3.22 ± 0.07a 1.43 ± 0.24 23.43 ± 1.92a

4.12 ± 0.24 3.64 ± 0.28a 3.24 ± 0.21ab 2.52 ± 0.24a 2.02 ± 0.25 1.91 ± 0.24 1.35 ± 0.08ab 3.32 ± 0.10ab 1.53 ± 0.18 23.65 ± 2.11a

4.28 ± 0.26 3.71 ± 0.14a 3.37 ± 0.26ab 2.58 ± 0.25ab 2.18 ± 0.27 2.09 ± 0.30 1.34 ± 0.06ab 3.43 ± 0.06bc 1.68 ± 0.12 24.66 ± 1.66ab

4.31 ± 0.32 3.65 ± 0.15a 3.38 ± 0.28ab 2.65 ± 0.25ab 2.18 ± 0.31 2.10 ± 024 1.44 ± 0.10bc 3.41 ± 0.04bc 1.60 ± 0.11 24.72 ± 2.05ab

4.32 ± 0.25 3.78 ± 0.28ab 3.46 ± 0.26bc 2.66 ± 0.27ab 2.16 ± 0.22 2.21 ± 0.13 1.45 ± 0.07bc 3.65 ± 0.07bc 1.57 ± 0.27 25.26 ± 1.06ab

4.72 ± 0.26 4.13 ± 0.14b 3.81 ± 0.24c 3.03 ± 0.27c 2.45 ± 0.29 2.22 ± 0.26 1.45 ± 0.05bc 3.71 ± 0.12c 1.55 ± 0.10 27.07 ± 1.92b

4.61 ± 0.26bc 4.15 ± 0.25b 3.47 ± 0.20ab 2.86 ± 0.22a 2.44 ± 0.10 2.33 ± 0.23 1.62 ± 0.13cd 3.70 ± 0.17c 1.32 ± 0.09 26.30 ± 1.10b

5.63 ± 0.30a 3.53 ± 0.28ab 2.12 ± 0.28a 2.05 ± 0.21a 2.04 ± 0.18a 1.85 ± 0.09a 1.76 ± 0.08a 1.67 ± 0.08a 43.91 ± 1.74a

5.61 ± 0.26a 3.13 ± 0.28a 2.06 ± 0.24a 2.05 ± 0.14a 2.53 ± 1.19b 2.10 ± 0.08b 1.94 ± 0.09ab 1.67 ± 0.10a 44.52 ± 1.62ab

5.66 ± 0.26a 3.58 ± 0.23ab 2.15 ± 0.28ab 2.17 ± 0.12a 1.99 ± 0.12a 1.96 ± 0.12ab 2.06 ± 0.16b 1.81 ± 0.10ab 45.03 ± 1.62ab

6.45 ± 0.20bcd 3.75 ± 0.25bc 2.28 ± 0.29a 2.32 ± 0.14ab 2.18 ± 0.10a 2.06 ± 0.10b 2.04 ± 0.13b 1.76 ± 0.04ab 47.50 ± 1.54bc

6.31 ± 0.27b 3.75 ± 0.30bc 2.32 ± 0.25a 2.35 ± 0.16ab 2.13 ± 0.14a 2.42 ± 0.10c 1.79 ± 0.08a 1.68 ± 0.03a 47.47 ± 2.13bc

6.40 ± 0.30bc 3.96 ± 0.26bc 2.47 ± 0.29a 2.39 ± 0.19ab 2.26 ± 0.14a 2.56 ± 0.09c 1.90 ± 0.19ab 2.09 ± 0.11c 49.29 ± 1.72c

6.87 ± 0.29cd 4.14 ± 0.25c 2.57 ± 0.34a 2.65 ± 0.09c 2.18 ± 0.16a 3.11 ± 0.07e 2.10 ± 0.07b 1.85 ± 0.17ab 52.54 ± 2.18d

6.95 ± 0.30d 5.13 ± 0.27d 3.34 ± 0.36b 3.24 ± 0.16d 2.26 ± 0.19a 2.79 ± 0.10d 2.05 ± 0.16b 1.90 ± 0.14b 53.96 ± 1.87d

Abbreviations: EAA, essential amino acid; NEAA, nonessential amino acid; TAA, total content of amino acid. Different superscript letters within rows represent significant differences (P < 0.05).

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weight. Changes in the contents of valine and isoleucine exhibited a pattern of ‘high-low-high’. A correlation between the essential amino acid content (except valine, isoleucine and methionine) and the total amino acid content was evident. There was a similar trend in NEAAs during embryonic development. The mean proportional amount of glutamic acid content was highest at 6.24% of dry weight. Tyrosine and proline contents were the lowest (1.96%, 1.80% of dry weight, respectively). The correlation between the NEAAs (except Ser and Pro) and the total amino acids was also evident. In addition, NH4Cl was determined in our experiment. A comparatively small amount was detected in the fertilized egg stage and cleavage stage. Content of NH4Cl increased to 3.52% of dry weight in the blastula stage and 4.67% of dry weight in the zoea stage.

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3.4. Fatty acid content The fatty acid composition (% of total) of different embryonic development stages of M. rosenbergii is presented in Table 3. Most important fatty acids in quantity were the saturates (SFA) 16:0, 18:0 and 14:0, the monounsaturates (MUFA) C18:1n-9c, C18:1n-9t, C16:1, C17:1, and the polyunsaturates (PUFA) C18:2n6, C18:3n-3, C20:2, C20:4n-6 (ARA), C20:5n-3 (EPA) and C22:6n-3 (DHA). Proportional contents of C16:0, C18:1n-9, C18:2n-6, C18:0 and C16:1 were high during the embryonic development stages. Contents of C16:0, C18:1n-9c, EPA and DHA decreased, while C14:0, 17:0, C24:0, C17:1 and ARA increased. The monounsaturates (MUFA) were utilized at a higher rate (7.56% of utilization) than the polyunsaturates (PUFA) (1.58%). Contents of SFA

Table 3 Fatty acid composition (%, of total fatty acids) during the embryonic development of Macrobrachium rosenbergii Fatty acid

C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 C24:0 ΣSAF C14:1 C15:1 C16:1 C17:1 C18:1n-9c C18:1n-9t C20:1n-9 C24:1n-9 ΣMUFA C20:2 C22:2 C18:2n-6 C18:3n-3 C20:3n-6 C20:4n-6 (ARA) C20:5n-3(EPA) C22:6n-3 (DHA) ΣPUFA

Embryonic development stages I

II

III

IV

V

VI

VII

VIII

1.81 ± 0.22b 0.47 ± 0.11a 23.4 ± 1.36c 1.18 ± 0.22bcd 6.54 ± 0.10c 0.32 ± 0.02a 0.22 ± 0.04 0.48 ± 0.12 0.14 ± 0.03 0.60 ± 0.09 35.06 ± 2.44c 0.16 ± 0.02 0.22 ± 0.02 8.47 ± 1.24ab 0.60 ± 0.10c 29.65 ± 1.54c 5.37 ± 1.03 0.40 ± 0.05a 0.11 ± 0.02 44.98 ± 2.21d 0.88 ± 0.09a 0.12 ± 0.03 9.13 ± 0.91bc 0.94 ± 0.94b 0.94 ± 0.11 0.15 ± 0.03a

1.54 ± 0.20ab 0.64 ± 0.14ab 22.11 ± 1.51abc 0.88 ± 0.14a 5.67 ± 0.11a 0.27 ± 0.02a 0.29 ± 0.03 0.77 ± 0.09 – 0.41 ± 0.04 32.58 ± 1.58abc 0.22 ± 0.03 0.39 ± 0.02 9.45 ± 0.56bc 0.24 ± 0.04a 26.64 ± 2.25bc 4.72 ± 1.24 0.75 ± 0.12bc – 42.41 ± 1.50cd 0.94 ± 0.07a 0.27 ± 0.03 8.50 ± 0.58ab 1.08 ± 0.04c – 1.49 ± 0.09d

1.68 ± 0.21ab 0.73 ± 0.12bc 23.12 ± 0.90bc 1.03 ± 0.16abc 6.26 ± 0.09b 0.29 ± 0.04a – – – – 33.11 ± 1.83bc – 0.33 ± 0.03 10.75 ± 1.18c 0.28 ± 0.06a 27.33 ± 1.84bc 5.33 ± 0.86 0.89 ± 0.17c – 44.91 ± 1.23d 0.92 ± 0.06a – 8.82 ± 0.92abc 1.02 ± 0.04bc – 1.29 ± 0.08c

1.41 ± 0.22ab 0.80 ± 0.07bc 19.79 ± 1.25a 1.27 ± 0.12cd 5.57 ± 0.13a 0.28 ± 0.03a 0.27 ± 0.04 0.22 ± 0.03 – – 29.61 ± 2.25a 0.15 ± 0.02 0.49 ± 0.13 9.08 ± 1.07abc 0.42 ± 0.04b 25.55 ± 2.10ab 4.16 ± 0.87 0.89 ± 0.09c 0.14 ± 0.01 40.88 ± 1.46bc 1.28 ± 0.14c 0.34 ± 0.04 7.63 ± 0.04a 1.09 ± 0.12c 0.44 ± 0.12 1.82 ± 0.07e

1.54 ± 0.19ab 0.64 ± 0.12ab 20.84 ± 1.50ab 0.97 ± 0.14ab 6.46 ± 0.12bc 0.27 ± 0.04a – – – 0.65 ± 0.04 31.37 ± 1.49ab – 0.44 ± 0.04 7.29 ± 1.08a 0.25 ± 0.03a 27.33 ± 1.74bc 5.47 ± 0.89 0.84 ± 0.11bc – 41.62 ± 1.36bc 0.97 ± 0.04ab – 10.21 ± 0.07c 1.03 ± 0.03bc 0.46 ± 0.06 1.12 ± 0.11b

1.28 ± 0.14a 0.85 ± 0.12bc 19.83 ± 1.62a 1.35 ± 0.11d 6.29 ± 0.14b 0.47 ± 0.05b 0.21 ± 0.04 0.39 ± 0.03 – – 30.67 ± 0.89ab 0.15 ± 0.02 0.38 ± 0.04 8.32 ± 1.15ab 0.40 ± 0.04b 24.45 ± 0.78ab 4.31 ± 1.12 0.79 ± 0.09bc 0.14 ± 0.03 38.94 ± 1.66ab 1.52 ± 0.09d 0.32 ± 0.03 10.19 ± 0.08c 1.24 ± 0.07d – 1.85 ± 0.13e

1.45 ± 0.22ab 0.88 ± 0.08c 21.07 ± 0.93abc 1.40 ± 0.16d 7.06 ± 0.08d 0.55 ± 0.11b – – – – 32.41 ± 1.52abc – 0.33 ± 0.03 8.35 ± 1.22ab 0.41 ± 0.04b 25.47 ± 1.28ab 5.17 ± 0.10 0.67 ± 0.10b – 40.40 ± 1.72bc 1.12 ± 0.07b – 8.42 ± 1.06ab 0.82 ± 0.02a 0.38 ± 0.03 1.51 ± 0.05d

1.52 ± 0.24ab 0.87 ± 0.09c 20.41 ± 1.00a 1.41 ± 0.15d 6.34 ± 0.10c 0.66 ± 0.05c – – – 0.89 ± 0.08 32.10 ± 1.38abc – – 8.09 ± 1.06ab 0.47 ± 0.07b 23.09 ± 1.27a 5.05 ± 0.09 0.72 ± 0.11bc – 37.42 ± 1.52a 1.03 ± 0.08ab – 8.21 ± 1.07ab 1.01 ± 0.06bc – 1.40 ± 0.08cd

1.62 ± 0.06e 0.92 ± 0.04e

1.62 ± 0.03e 0.88 ± 0.11de

1.22 ± 0.04cd 0.89 ± 0.07de

1.30 ± 0.13c 0.79 ± 0.07cd

1.26 ± 0.06cd 0.72 ± 0.05bc

1.16 ± 0.09bc 0.62 ± 0.03b

1.04 ± 0.06ab 0.48 ± 0.06a

1.00 ± 0.04a 0.47 ± 0.05a

14.70 ± 0.85ab

14.78 ± 1.24ab

14.16 ± 1.27ab

14.69 ± 0.46ab

15.77 ± 0.70bc 16.90 ± 1.21c

13.77 ± 0.50a

13.12 ± 0.40a

Denotation for fatty acid, C: an-b, C: the number of carbon atoms; a: the number of double bonds; b: the position of the first double bond from the alkyl; ΣSFA: the summation of saturated fatty acid; ΣMUFA: the summation of single unsaturated fatty acid; ΣPUFA: the summation of polyunsaturated fatty acid; “–” : means unfound. Different superscript letters within rows represent significant differences (P < 0.05).

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Table 4 Mean digestive enzyme activities during the different stages of embryonic development of M. rosenbergii (X ± SD, n = 3); Unit: U/egg Stage Fertilized egg (I) Cleavage (II) Blastula (III) Gastrula (IV) Egg nauplius (V) Egg metanauplius (VI) Protozoea (VII) Zoea (VIII)

Pepsin (U/egg) −2

Trypsin (U/egg) d

13.51 × 10 ± 0.007 16.26 × 10− 2 ± 0.011e 7.69 × 10− 2 ± 0.010b 4.00 × 10− 2 ± 0.007a 9.42 × 10− 2 ± 0.003bc 11.20 × 10− 2 ± 0.010c 35.43 × 10− 2 ± 0.014f 58.17 × 10− 2 ± 0.014g

−2

Amylase (U/egg) c

8.81 × 10 ± 0.008 6.86 × 10− 2 ± 0.008a 3.95 × 10− 2 ± 0.005ab 2.28 × 10− 2 ± 0.008ab 4.81 × 10− 2 ± 0.006b 5.16 × 10− 2 ± 0.006b 23.11 × 10− 2 ± 0.032d 27.40 × 10− 2 ± 0.045e

−2

Cellulase (U/egg) c

34.48 × 10 ± 0.036 20.98 × 10− 2 ± 0.015ab 26.51 × 10− 2 ± 0.076abc 17.19 × 10− 2 ± 0.042a 24.37 × 10− 2 ± 0.059abc 26.56 × 10− 2 ± 0.110abc 32.30 × 10− 2 ± 0.065bc 55.27 × 10− 2 ± 0.035d

−2

Lipase (U/egg) bc

3.66 × 10 ± 0.008 2.07 × 10− 2 ± 0.008a 2.10 × 10− 2 ± 0.007a 1.93 × 10− 2 ± 0.006a 2.23 × 10− 2 ± 0.118ab 2.95 × 10− 2 ± 0.006abc 3.59 × 10− 2 ± 0.010bc 3.75 × 10− 2 ± 0.004c

0.52 × 10− 3 ± 0.0009b 0.26 × 10− 3 ± 0.0009a 0.52 × 10− 3 ± 0.0009b 0.48 × 10− 3 ± 0.0010b 0.19 × 10− 3 ± 0.0010a 0.30 × 10− 3 ± 0.0010a 0.34 × 10− 3 ± 0.0011ab 0.19 × 10− 3 ± 0.0008a

Different superscript letters within vertical rows represent significant differences (P < 0.05).

decreased from 35.06% to 32.10%. As to the utilization of individual fatty acids, there was a preferable metabolism of C18:1n-9, C16:0 and C18:2n-6. There was a significant decrease in both ∑SAF and ∑MUFA in the gastrula stage, but contents of both of these fatty acids increased after this stage. Higher metabolism of n-3 fatty acids was detected. Both DHA and EPA were catabolized, with DHA being more depleted than EPA. 3.5. Digestive enzyme activity There were two patterns of variation in digestive enzyme activities during the embryonic development of M. rosenbergii (Table 4). Activities of lipase decreased while activities of pepsin, trypsin, amylase and cellulase exhibited a pattern of ‘high-low-high’. Digestive enzyme activities were relatively high in the fertilized egg stage. In the fertilized egg stage, the activity of pepsin was 13.51 × 10− 2 ± 0.007 U/egg, decreased to 4.00 × 10− 2 ± 0.007 U/egg in the gastrula stage, and then rose to 58.17 × 10− 2 ± 0.014 U/egg in

the zoea stage. The activity of trypsin was 8.81 × 10− 2 ± 0.008 U/egg in the fertilized egg stage, decreased to 6.86 × 10− 2 ± 0.008 U/egg in the cleavage stage, then increased to 27.40 × 10− 2 ± 0.045 U/egg in the zoea stage. Amylase activity was 34.48 × 10− 2 ± 0.036 U/egg in the fertilized egg stage, dropped to 17.19 × 10− 2 ± 0.042 U/egg in the gastrula stage, and then increased to 55.27 × 10− 2 ± 0.035 U/egg in the zoea stage. Cellulase activity was 3.66 × 10− 2 ± 0.008 U/egg in the fertilized egg stage, decreased to 1.93 × 10− 2 ± 0.006 U/egg in the gastrula stage, and then increased gradually to 3.75 × 10− 2 ± 0.004 U/egg in the zoea stage. Lipase activity was 0.52 × 10− 3 ± 0.0009 U/egg in the fertilized egg stage and decreased to 0.19 × 10− 3 ± 0.0008 U/egg in the zoea stage. During the embryonic development, activities of pepsin, trypsin, amylase and cellulase decreased to the lowest point in the gastrula stage, then increased quickly in the following egg nauplius stage. Trypsin activity decreased from the fertilized egg to cleavage stage, but pepsin activity increased. Activities of amylase, cellulase

Fig. 2. Morphological structure of several stages in the eggs of Macrobrachium rosenbergii during development. 1. Fertilized egg stage, 2. Gastrula stage, 3. Protozoea stage, 4. Zoea stage. ME: first egg membrane; YK: yolk; OA: organ anlage; CEA: compound eye anlage.

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and lipase decreased in the cleavage stage. Lipase activity decreased after the gastrula stage, but activities of the other enzymes increased gradually to their highest levels during the zoea stage. 3.6. Morphogenesis Some morphological photographs are presented in Fig. 2. During the fertilized egg stage, the egg was full of yolk granules that were homogeneously distributed. Some organ anlages such as the thoracico-abdominal fold could be observed during gastrula stage. During the protozoea stage, the compound eye anlage can be observed and the yolk granules near organ anlages were much different from those observed in the fertilized egg stage. During zoea stage, the development of the brain, heart, compound eyes and appendages was noted. Moreover, some yolk granules still could be observed during late zoea stage. 4. Discussion Egg size appears to be species-specific among decapods. The size of eggs correlates with stage of development and serves as an indicator of energy content (Herring, 1974). Generally, species with larger size eggs contain more yolk nutrients and their embryonic development time is longer. On average, egg size of M. rosenbergii (0.081 mm3) is much smaller than that of Cherax quadricarinatus, Alpheus saxidomus (0.247 mm3) (Wehrtmann and Graeve, 1998) and Nephrops norvegicus (1.46 mm3) (Rosa et al., 2003), but is larger than that of Palaemonetes schmitti (0.056 mm3) (Wehrtmann and Graeve, 1998). It seems that egg volume relates little to lipid content. Egg volume of M. rosenbergii is one-third of A. saxidomus, one-eighteenths of N. norvegicus, but larger than P. schmitti. However, the eggs of M. rosenbergii contained more lipid (30%, average) than did A.saxidomus, P. schmitti, Nauticaria mateiianice, Betaeus emarginatus and N. norvegicus (<20%, dry weight) (Wehrtmann and Graeve, 1998; Rosa et al., 2003). Higher lipid and protein content in the eggs of M. rosenbergii might be the reason that there are four relatively long larval stages in the embryonic development of this species, including egg nauplius, egg metanauplius, protozoea and zoea stage. Weight of eggs during zoea stage increased from that during fertilized stage, mainly because of the increase of water content. Water provided a liquid environment for embryo and the higher water pressure during zoea stage might be cause of the embryo breaking though the egg membrane in preparation for hatching.

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During embryonic development of M. rosenbergii, digestive enzymes hydrolyze the yolk to provide energy for the development of organs and systems in the embryo. Pepsin, trypsin and amylase activities were determined relatively high in larvae of Penaeus chinensis, Penaeus japonicus, Eriocheir sinensis and Portunus trituberculatus (Pan and Wang, 1997). Trypsin plays a very important role in larvae of Penaeus vannamei in comparison to pepsin (Wang et al., 2004). Digestive enzymes in M. rosenbergii exist before the larvae start to feed (Biesiot, 1986), and also during every stage of embryonic development according to the results of our investigation. Activities of pepsin, trypsin, amylase, cellulase and lipase remained at a certain level during the fertilized egg stage. The present study indicates that carbohydrate is the main energy source in the early stage of embryonic development. Lipid also served as an energy source, and protein served mainly as the structural substance. Yolk proteins were hydrolyzed into amino acids by pepsin and trypsin, and were presumably turned into structural components in the embryo. A high protein content has also been found in the embryos of E. sinensis (Tian et al., 2003) and Homarus americanus (Biesiot, 1986). These results indicate that high activities of pepsin and trypsin played an important role in the utilization of proteins in the yolk. In contract to larvae of Scylla serrata (Tang et al., 1995) and P. trituberculatus (Pan and Wang, 1997), Pepsin activity was higher than that of trypsin in eggs of M. rosenbergii, that pepsin is principally responsible for the hydrolysis of yolk protein. The low enzyme activity of lipase might be reflective of a relatively long embryonic development time (about 480 h) of M. rosenbergii. During gastrula stage, organ anlage appeared. Some anlages, such as those of the optic lobe, the ventral plate and thoracic-abdominal fold formed (Zhao et al., 1998a). However, the lowest enzyme activities of pepsin, trypsin, amylase and cellulase at this stage might be due to the gradual depletion of the ovum-originated mRNA, followed by the expression of mRNA by the embryo to synthesize more digestive enzymes to ensure the utilization of yolk. After this stage, those digestive enzyme activities increased gradually. High activities of pepsin, trypsin, amylase and cellulase in the zoea stage may be prepare for hatching and the first molt, as more energy will be needed at that time. During the zoea stage, yolk was hydrolyzed at a high rate and embryological organs were nearly developed, leading to the beginning of physiological functions. For example, the heart began to beat and blood started to circulate in the embryo. Proteins in yolk acted mainly as

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an energy source at this time, resulting in a decrease in the content of protein at this stage. The larvae of M. rosenbergii are carnivorous and activities of pepsin and trypsin increased during the zoea stage in preparation for larval feeding. According to studies with Penaeus setiferus (Lovett and Felder, 1990) and Penaeus monodon (Fang and Lee, 1992), a high enzyme activity of amylase was independent of feeding. It was believed that high activity of amylase was due to low carbohydrate levels (Rodriguez et al., 1994). The relationship between amylase activity and carbohydrate in embryos of decapods requires additional study. It has been revealed that amino acids (Litaay et al., 2001), monoscacharides (Monroy and Tollis, 1961) and nucleosides (Schneider and Whitten, 1987) could be transported into the embryos of marine invertebrates. Rosa et al. (2003) assumed that embryos of N. norvegicus could absorb some compounds. In the present study, the content of essential amino acids increased in eggs, may be the result of organic compounds could be transported into embryos of M. rosenbergii. The ratio of content of each amino acid (EAA) to total amino acids (TAA) remained unchanged during different embryonic development stages, which contributed to the completion of the embryonic development of M. rosenbergii. The increase in TAA are indicative of the fact that protein in yolk acts as the main structural substance during embryonic development of M. rosenbergii. Increase in TAA were also determined in eggs of N. norvegicus (Rosa et al., 2003). Quantitatively, the most important amino acids were leucine, lysine, valine, arginine, glutamic acid and aspartic acid during the embryonic development of M. rosenbergii. Regarding the function of single amino acid, leucine is a ketone-producing amino acid. It could be transformed into acetyl-CoA and acetyl-acetic acid, which are important intermediates in carbohydrate and lipid metabolism (Shen and Wang, 1990). Arginine was proven to be crucial in energy metabolism by maintaining glycolysis under hypoxic conditions (Gade and Grieshaber, 1986). Valine is a carbohydrate-producing amino acid and may be associated with carbohydrate metabolism through citric acid cycle. Content of glutamic acid was high during embryonic development, which may have resulted from nitrogen metabolism in eggs of M. rosenbergii. Glutamic acid turned into glutamine, which is deaminated to produce NH3 (Shen and Wang, 1990). NH3 can be excreted along with Cl−. An increase in the content of NH4Cl after the blastula stage also suggests that NH4+ and Cl− are being excreted

together. Aspartic acid can be synthesized from other amino acids and carbohydrate. Tyrosine can be used to synthesize melanin (Shen and Wang, 1990), which plays a central role in the accumulation of compound eye pigments. High content of these amino acids is closely correlated with their important role in the embryo. The consumption pattern of different fatty acids in eggs of M. rosenbergii during the embryonic development did not differ markedly from that of other crustaceans (Wehrtmann and Graeve, 1998; Wehrtmann and Kattner, 1998; Morais et al., 2002; Li et al., 2003; Rosa et al., 2003). The most important fatty acids were C16:0, C18:0, C16:1, C18:1n-9 and C18:2n-6 in eggs of M. rosenbergii. Variation of fatty acid content was closely associated with morphological variation. Content of C18:1n-9 decreased significantly in the cleavage stage, which suggests that this fatty acid was involved in some important metabolic functions during the cleavage stage and probably serves as a main energy source during embryonic development. During early stages (from fertilized egg to gastrula stage), SFA were utilized preferentially. C16:0 may be main energy source. As anlages appeared during gastrula stage, the proportional amounts of SFA and MUFA were detected to decrease much from the blastula to gastrula stages. C14:0, C16:0, C18:0 C16:1 and C18:1n-9 presumably provided much energy for the formation of anlages. Appendage alange formed during egg nauplius and egg metanauplius stage (Zhao et al., 1998a), and MUFA were preferentially used for energetic purposes and C18:1n-9 was still the main energy source. During protozoea stage, compound eyes formed and brain and heart anlages came into being. In the zoea stage, the compound eyes became larger, the blood started to circulate and the heart beats at about 200 times per minutes (Zhao et al., 1998a,b). PUFA acted as main energy source during these two stages. C20:2, C18:2n6, C18:3n-3, EPA and DHA presumably played an important energetic role during late embryonic development stages. That rapid decrease of total lipids in the zoea stage was closely associated with the formation and development of many organ anlages. The zoea stage of M. rosenbergii lasted about 90 h and more energy was needed. So some fatty acids were probably used in the synthesis of organs during this stage. UFA were metabolized at a higher rate than SFA, with MUFA being preferentially used for energetic purposes in N. norvegicus (Rosa et al., 2003). An increase in fatty acids content such as C18:3n-3 and

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ARA indicated that they were assimilated gradually into the components of the embryo. ARA and EPA are important structural components of cell membranes and also the precursors of prostaglandins (Lilly and Bottino, 1981). DHA and EPA were detected in relatively low proportion in eggs of M. rosenbergii. In general, C22:6n-3 has been considered as one of the important fatty acids in decapod eggs, accounting for roughly 10–20% of the total fatty acids (Kattner et al., 1994; Wehrtmann and Kattner, 1998). We found that the contents of both EPA and DHA decreased during the embryonic development, and DHA that was more depleted than EPA. EPA and DHA played an important role to improve the hatchability of crustacean (Xu et al., 1994; Cavalli et al., 1999). Eggs still contain some yolk when they hatch. As the first larval stage of M. rosenbergii is a non-feeding stage, the remaining nutriment favors their independence of external energy resources when external feeding begins and would increase the chances for the first successful molt. 5. Conclusion Biochemical changes and digestive enzyme activities were found to reflect changes in morphogenesis during the embryonic development of M. rosenbergii. We have demonstrated the following points: Firstly, Five digestive enzyme activities remain at a certain level during fertilized egg stage and carbohydrate was the main energy source in early stage of embryonic development. Secondly, gastrula stage is an important stage for organ anlage come into being. Thirdly, The ratio of EAA to TAA remained unchanged in different embryonic development stages, which ensured the completion of embryonic development. Lipid in yolk acted mainly as an energy source and the most important fatty acids were C16:0, C18:0, C16:1, C18:1n-9 and C18:2n-6 in eggs of M. rosenbergii. SFA acted as main energy source during early stages from fertilized egg to gastrula stage, and MUFA acted as main energy source from egg nauplius to egg metanauplius stage. HUFA provided mainly energy during late stages. Fourthly, eggs still contain some yolk till hatch, which ensures the first successful molt and favors their independence of external energy resources when external feeding begins. Acknowledgements Sincere thanks are forwarded to Zhu Yuezhong, Li Xuelin and all their co-workers of the Flourishing Well-

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