Indian J. Fish., 45(2) : 141-148, Apr.-Jun., 1998. Fatty acid profile during embryonic development of the cultivable freshwater prawn. Macrobrachium ...
Indian J. Fish., 45(2) : 141-148, Apr.-Jun., 1998
Fatty acid profile during embryonic development of the cultivable freshwater prawn Macrobrachium malcolmsonii (H. Milne Edwards) M. J O H N SAMUEL, T. KANNUPANDI AND P. SOUNDARAPANDIAN Centre of Advanced Study in Marine Biology, Parangipettai - 608 502, India ABSTRACT The fatty acid profile during embryonic development of Macrobrachium malcolmsonii was studied. Docosahexaenoic acid (DHA) was the highest (12.82 %) in the mature ovary but it decreased linearly from 12.77 % in the initial stage (0 hour) to 9.79 % in the final stage (312 hour). Eicosapentaenoic acid in embryo showed inverse linear relationship with time after fertilization. Similar trend was also observed for 22:5n 6 and 22:6n 3 fatty acid contents with time. Saturates in general increased during stage II and III. However, during stage IV, 12.51 % was utilised. Stage II utilised 7.42 % monounsaturates, a high percentage when compared with other stages (stage I, 0.3 %, stage III, 3.84 % and stage IV, -4.86 %). It is interesting to note that among polyunsaturates (PUFA), 20:4n 6 and 20:5/i 3 were utilised more in stages I, III, and IV than in stage II. However, other fatty acids (20:2n 6,9 and 22:5n 6) showed not much difference in utilization. In general, PUFA were utilised more in stage III (7.40 %) and stage IV (0.05 %) than in stage I (1.36 %) and stage II (0.63 %).
Introduction Suppressed protein utilization and enhanced lipid metabolism are characteristics of cleidoic eggs, a feature found also in m a n y c r u s t a c e a n species (Pandian, 1972; Pillai and Subramoniam, 1985). The principal components of most lipids are fatty acids (Castell, 1981). Although a number of studies are available on the fatty acid changes during embryogenesis for other invertebrates, only limited information is available for prawns. Two of the main fatty
acids in the eggs and embryos, 20:5n 3 and 22:6n 3 are regarded as nutritionally essential for most of the marine species (Kanazawa etal., 1979; Langdon and Waldock, 1981; Watanabe, 1982; Levine and Sulkin, 1984). Though the energy requirement is met from the oxidation of fat during embryonic deve -lopment of M. malcolmsonii (Mathavan et al., 1986), the relative proportion of fatty acids accompanying embryogenesis is still unknown and hence this study.
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Materials and m e t h o d s
Results
Mature males (length 155 mm and weight 33 g) and equal number of mature females (length 150 mm and weight 31.4 g) of M. malcolmsonii were collected from the tanks of Manampadi (11° 29'N and 79° 46'E), Tamil Nadu. They were acclimatised to laboratory conditions (salinity: 0.5 ppt, temperature: 28 ± 2°C, DO: 5 ppm and photophase: 12/12 h L/D) and maintained in a 180 x 60 cm fibreglass tank. One third of the water was changed daily and the prawns were fed with clam meat. The females were carefully examined for premating moult. As soon as the females experienced premating moult, mating was allowed by introducing a newly pre-mating moulted female with a mature male in 1:1 ratio in a 50 1 fibreglass tank. Soon after mating, the male was separated and the gravid female was reared in sea water with 6 ppt salinity. The developing embryos of 0 (stage I), 72 (stage II), 120 (stage II), 192 (stage III) and 312 (stage IV) hours were identified following Rao (1986) using a stage binocular microscope (Meiji, Labex, Japan). Similarly, the mature ovary was also dissected out immediately from the premating moulted female. Both the mature ovary and the developing embryos were used for fatty acid analyses.
Values for fatty acid profile in mature ovary and the developing embryos (0, 72, 120, 192 and 312 hours) are given in Table 1. Docosahexaenoic acid (22:6n 2) was higher (12.82 %) in the mature ovary but it decreased linearly from 12.77 % in the initial stage (0 hour) to 9.79 % in final stage (IV stage) (r2 = 0.9647). Though very low 13.0 fatty acid content (0.14 %) was observed in the mature ovary, it increased linearly from 0 hour (0.21 %) to 312 hour (1.67 %) embryo (r2 = 0.9583). Eicosapentaenoic acid (20:5n. 3) increased from 12.32 % in mature ovary to 12.84 % in eggs immediately after fertilization (0 hour), whereas, from 0-312 hour embryo showed inverse linear relationship with time. Similar trend was also observed for 22:5n 6 and 20:2n 6,9 fatty acid contents with time.
The fatty acid methyl esters of the samples were injected into the gas chromatography (HP 5890) capillary column coated with 5 % phenyl silicane at a temperature from 170°C to 310°C for 23.33 minutes. Flame Ionization Detector was used for the analysis. Based on the retention time the different fatty acids of the samples were identified. Using ABSTAT 3.01 statistical package, correlation and regression were computed.
An increase in 16: In 7 fatty acid content in 120 hour embryo was noticed. However, 16:0 fatty acid increased in 120 and 192 hour embryo than in other stages. No significant trends were shown by U:ln 5, 15:0, 15:l/i 6, 16:0, 1 6 : 1 K 7, 17:0, 17:1, 17:In 8, 18:0, 18:Ire 7, 18:ln 9, 19:1 and 20:0 fatty acids with time after fertilization. But fatty acids 12:0, 18:1, 18:3n 6,9,12, 20:2n 6,9, 13:0, 14:0, 16:ln 6,9, 20:4n 6, 20:5n 3, 22:5?i 6, 22:6n 3 significantly correlate with time after fertilization. The percentage of saturates 16:0 (r2 = 0.4965), 18:0 (r2 = 0.3027) and 20:0 (r 2 = 0.2309) showed not much difference among different stages after fertilization and they showed very little correlation with time and it was nonsignificant. In general, the saturated fatty acids showed very little correlation with time and it was not significant (r2 = 0.1706; P > 0.05). Mono unsaturates showed an inverse linear
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Fatty acid profile during embryonic development in a prawn TABLE 1. Fatty acid profile during embryonic development of Fatty acid
M. malcolmsonii
% of fatty acid Hours after fertilization
Ovary * 0(SI)
*72(SI)
*120(SII)
:l
192(SIII)
*312(SIV)
12:0
0.46
0.56
0.57
0.62
1.12
1.53
13:0
0.14
0.21
0.20
0.59
1.01
1.67 4.45
14:0
2.83
7.30
7.19
5.97
5.49
14: Ire 5
-
0.34
0.22
-
-
15:0
1.77
1.72
1.66
2.41
3.56
15:ln 6
-
0.29
0.21
-
-
8.52
16:0
2.76
10.67
10.24
10.14
13.20
13.28
16:ln 5
0.17
0.21
0.25
0.30
0.36
0.40
16: Ire 7
9.29
12.37
12.27
13.71
4.51
3.91
17:0
2.73
4.21
3.45
1.80
2.95
0.22
17:1
1.71
2.41
2.42
2.39
2.52
4.68
17:ln 8
1.70
1.98
1.51
0.21
0.73
0.53
18:0
3.36
4.43
4.43
4.06
7.81
4.65
18:1
0.64
0.92
0.97
1.36
1.42
1.66
18:ln 7
-
2.29
3.81
3.72
3.88
3.91
18:ln 9
10.44
8.28
7.91
2.37
1.09
0.98
8.26
4.21
4.98
6.09
13.29
21.21
18:2n 6,9 18:3n 6,9,12
1.10
0.48
0.32
0.59
1.96
2.91
19:1
3.19
4.89
4.11
2.20
7.91
11.21
20:0
0.33
0.42
0.15
0.28
1.41
0.32
1.88
0.52
0.26
0.95
2.78
2.49
20:4n 6
12.24
5.66
5.03
4.99
4.08
3.43
20:5n 3 •
12.32
12.84
12.24
11.89
10.24
7.61
22:5re 6
1.08
4.52
4.21
3.82
2.92
2.61
22:6n 3
12.82
12.77
12.63
12.01
10.86
9.79
Saturated
22.29
29.09
27.79
28.93
36.63
24.12
Monounsaturated
27.14
33.98
33.68
26.26
22.42
27.28
33.87 .
28.82
20:2re 6,9
41.90 41.27 43.26 48.77 Polyunsaturated SI - Stage I; SII - Stage II; SIII - Stage III; SIV - Stage IV.
relationship with time, and it was significant (r2 = 0.6703; P < 0.05). Similarly, PUFA also showed such trend with time wich was significant (r2 = 0.9298; P < 0.05). The percentage of fatty acid utilization is reported in Table 2. Saturates in general increased during stage II and
III. However, during stage IV, 12.51 % was utilised. Stage II utilised 7.42 % monounsaturates, a high percentage when compared with other stages (stage I, 0.30 %; stage III, 3.84 % and stage IV, -4.86 %). It is also interesting to note that among PUFA, 20:4ra 6 and 20:5n 3 were utilised more in stages I, III and
M. John Samuel et al.
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TABLE 2. Fatty acid utilization during embryonic development of M. malcolmsonii Fatty acid
% of fatty acid utilized Stage I Stage II Stage III Stage IV
12:0
-0,01
-0.05
-0.5
-0.41
13:0
0.01
-0.39
-0.42
-0.66
14:0
0.11
1.22
0.48
1.04
14:1/7 5
0.12
0.22
15:0
0.06
-0.75
-1.15
0.80
15:1/7 6
0.80
0.21
16:0
0.10
-3.06
-0.08
4.76
16:ln 5
-0.04
-0.05
-0.06
-0.04
16;ln 7
0.10
-1.44
9.20
0.60
17:0
0.76
1.65
-1.15
2.73
17:1
-0.01
0.03
-0.13
-2.16
17:l/i 8
0.47
18:0
1.30
-0.52
0.20
0.37
-3.75
3.16 -0.24
18:1
-0.05
-0.39
-0.06
18:l/i 7
-1.52
0.09
-0.16
-0.03
0.37
5.54
1.28
0.11
-0.77
-1.11
2.80
1.08 -0.95 -3.30
18:ln 9 18:2// 6,9 18:3n 6, 9,12
0.16
-0.27
-1.37
19:1
0.78
1.91
-5.71
20:0
0.27
-0.13
-1.13
1.09
20:2n. 6,9
0.29
0.61
1.36
0.26
20:4n 6
0.63
0.04
0.91
0.65
20:5/7. 3
0.60
0.35
1.65
2.63
22:5n 6
0.31
0.39
0.90
0.31
22:6/J 3
0.14
0.62
1.15
1.07
Saturated
1.30
-1.14
-7.70
12.51
Mono unsaturated
0.30
7.42
3.84
-4.86
Poly unsaturated
1.36
0.63
7.40
5.05
*Readings in negative show increase in fatty acid whereas others show fatty acid utilization: IV than in stage II. However, other fatty acids {20:2n 6,9 and 22:5n 6) showed not much difference in utilization. In general PUFA were found to be utilised
more in stage III (7.40 %) and stage IV (5.05 %) t h a n in stage I (1.36 %) and stage II (0.63 %). Discussion
The predominance of 22:6n 3, 20:5n 3 and 20:4n 6 fatty acids in the m a t u r e ovary of M. malcolmsonii was found to be on par with the observations of Middleditch et al. (1979, 1980) in P. setiferus, P. stylirostris and P. vannamei respectively. Since the percentage of PUFA (18:3n 3, 20:4rc 6, 20:5n 3 and 22:6M 3) in M. malcolmsonii ovary is more or less similar to t h a t of the ovaries of P. japonicus, P. indicus, P. setiferus, P. stylirostris and P. vannamei, it is possible to suggest t h a t the PUFA pattern among the prawn species both in freshwater and marine environment show a similar trend. This coincides with the observation of Castell (1979), that the PUFA pattern among the crustaceans in freshwater and marine environments tends to be the same. During embryogenesis however, with no exogenous supply, compositional changes could r e s u l t from either oxidative degradation of fatty acids as the source of energy, or less likely from endogenous synthesis (Whyte et al, 1991). Oxidative depletion of principal fatty acids 20:5n 3 and 22:6rc 3 implied no corresponding increase in two carbon higher isomers and it was evident t h a t both the elongase and desaturase activities are considered low in M. malcolmsonii. Such low elongase and desaturase activity was observed in bivalves (Waldock and Holland, 1984; Whyte et al, 1991, 1993). Higher PUFA utilization in stages I, III and IV rather t h a n in stage II in the present study can be attributed to the fact t h a t the developing eggs require enormous energy for cleavage, gastrulation and
Fatty acid profile during embryonic development in a prawn
cellular differentiation in early stages and organogenesis in the later developmental stages. These findings agree with Mathavan et al. (1986) that M. malcolmsonii eggs utilise 0.79 J/mg/day energy during early and late stages of development but only about 0.42 J/mg/ day energy in mid development, and S u b r a m o n i a m (1991) t h a t cellular differentiation in mole crab starts soon after gastrulation and requires enormous energy expenditure. Oxidative depletion of fatty acids 19:1, 20:4n 6, 20:5n 3 and 16:0, 17:0, 18:0, 20:5rc 3 in stage I and stage IV respectively stresses the importance of these acids as an energy.source for cleavage, gastrulation and cellular differentiation in stage I and organogenesis in stage IV respectively. Wide fluctuation of saturates, especially 16:0 suggests its importance during embryogenesis as an energy source in M. malcolmsonii. The constant amounts of 15:0, 12:0, 16:In 5, 17:ln 8 and 18:ln 7 fatty acids present t h r o u g h o u t e m b r y o g e n e s i s of M. malcolmsonii show their structural role as reported by Reichwald-Hacker et al. 1979), Whyte (1988) and Whyte et al. (1990) in bivalves. PUFA of both n -3 and n -6 types are important in biomembranes, particularly in the vascular and nervous systems (Crawford et al., 1989; Vergroesen, 1989). Lands (1986) h a s shown t h a t n 3 fatty acids act as a suppressant to the biosynthetic pathway of prostaglandins, while n -6 fatty acids enhance the pathway. The slow decrease in the arachidonic acid (20:4rc 6) in the present study may also be due to the biosynthesis of prostaglandins since, arachidonic acid is the precursor for the biosynthesis of prostaglandins (Middleditch et al, 1979) and these have structural roles in phospholipids and permeability (Ahlgren
145
et al., 1992). The declining percentage of C2() isomere of PUFA such as 20:4n 6 and 20:5n 3 during embryogenesis are also due to the enzymatic oxidation which yields pharmacologically active eicosanoids that play an active role in water transport and osmoregulation similar to the observation of Freas and Grollman (1980) in Modiolus demissus. Rao (1986) demonstrated the formation of pigmented eyes in stage III in M. malcolmsonii. Sargent et al. (1994) emphasised the essential role of DHA for the development of retina. Subsequently, their works also showed t h a t the deficiency of DHA leads to twilight vision in the marine fish larvae. Surprisingly, the higher utilization of DHA in stage III than in the other stages in the present study may be due to the formation of pigmented eyes. Smith (1952) and Ando (1962) believed that the triglyceroides are not utilised until the last stages of yolk utilization. Ando (1968) observed t h a t the triglyceroides begin to decrease at a steady rate from the time shortly after hatching. Hayes et al. (1973) also found no evidence to support the selective retention of triglyceroides in the yolk until prior final yolk absorption. The present study also shows less utilization of n -3 and n -6 PUFA t h a n monoenes, and saturates may be a conservative pattern which confirms t h e speculation, that M. malcolmsonii h a s an obligatory b u t passive m i g r a n t l a r v a l s t a g e (Kewalramani, 1973). Since these larvae (Zoea I) are transported to the estuary soon after hatching in water with low salinity, they would need sufficient energy to enhance their survival.
Acknowledgment Thanks are due to the Director,
M. John Samuel et al.
Centre of Advanced Study in Marine Biology a n d the A u t h o r i t i e s of A n n a m a l a i U n i v e r s i t y for p r o v i d i n g facilities. F i n a n c i a l a s s i s t a n c e b y T a m i l N a d u S t a t e Council for Science a n d Technology is gratefully a c k n o w l e d g e d .
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