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Seasonal variations in the biochemical composition and lipids of the digestive gland in the shrimp Pleoticus muelleri bate
Comp. Biochem. PhysioL Vol. 98B,No. 2/3, pp. 253-260, 1991 Printed in Great Britain
0305-0491/91 $3.00+ 0.00 © 1991PergamonPress pie
SEASONAL VARIATIONS IN THE BIOCHEMICAL COMPOSITION A N D LIPIDS OF THE DIGESTIVE GLAND IN THE SHRIMP PLEOTICUS MUELLERI BATE WALTERHORAClOJECKEL, JULIAELENAAIZPUNDE MORENOand VfCTORJORGE MORENO Instituto Nacional de Investigaci6n y Desarrollo Pesquero (INIDEP), C.C. 175 Playa Grande s/No. 7600, Mar del Plata, Argentina (Received 9 July 1990) Aimtract--l. Seasonal variations in the biochemical composition, lipid classes and fatty acids in the digestive gland of the shrimp Pleoticus muelleri from San Jorge Gulf, Argentina, were studied during 1984-1985. 2. The relative size of the digestive gland decreased significantlyin fully mature shrimps of both sexes during the summer. 3. Immature females and mature males gradually increased the level of total lipids from winter toward the autumn as a reflex of the annual cycle of feeding. 4. Total lipids fell markedly during the ovary maturation in summer whilst protein increased. Free fatty acids, triacylglycerolsand phospholipids were apparently transferred to the developing ovary; however, the immediate dietary supply of lipids and protein appeared to be preponderant from incipient maturation until the completion of ovary development. 5. Total lipids decreased with the maturation of the male reproductive system in spring with the suggestion of their utilization during spermatogenesis. 6. Maturing females appeared to use selectivelyfatty acids 16:0, 16:1 and 18:1 of neutral lipids during oogenesis in summer whereas males used the mentioned acids of polar lipids apparently due to metabolic stress. 7. The results are discussedin terms of concentration and absolute amounts in relation to physiological and ecological factors.
INTRODUCTION
MATERIALSAND METHODS
The digestive gland is generally regarded as a major storage organ in decapod crustaceans (Huggins and Munday, 1968; Allen, 1971; Guary et al., 1974; Teshima and Kanazawa, 1983). The study of the digestive gland is of considerable interest because of its roles in the accumulation and cyclic mobilization of reserves during the molting process (Travis, 1955; O'Connor and Gilbert, 1969; Galois, 1980; Jeckel et aL, 1990); the contribution with nutrients to the ovary during vitellogenesis (Armitage et al., 1973; Kulkarni and Nagabhushanam, 1979; Adiy0di, 1985); the mobilization of reserves during starvation (Heath and Barnes, 1970; Armitage et al., 1972) as well as in the digestion and absorption processes (Dall, 1981). The purpose of the present study was to characterize the seasonal pattern of the biochemical composition, lipid classes and fatty acids in the digestive gland of the marine shrimp Pleoticus muelleri, in relation to physiological factors (such as sex and stage of gonad maturation) and ecological factors (e.g. food quality and availability, and temperature). This paper complements previous works which reported the morphometric and biochemical changes in the reproductive system of the shrimp (Jeckel et al., 1989a, b). Hence, a great part of the results presented here are discussed in terms of concentration and absolute amounts in relation to the mentioned studies.
The organisms and the capture area (San Jorge Gulf, Argentina) as well as the biological and biochemical methods utilized in this study have been described in previous works (Jeekel et al., 1989a, b). The animals were separated according to the sex and stage of gonad maturation, and subsequently weighed. Because we were interested in general trends of each sex in the population studied, individual molting stages were ignored; however, shrimps with signs of imminent molt or recently molted were not used. In both sexes, Sl represents sexual immaturity; $2, incipient maturation; and $3, complete maturity. The male reproductive system referred to here comprised the set testes plus spermatophores. Digestive glands corresponding to each sex and stage of gonad maturation were carefully extracted from the shrimps inside an icebath. They were subsequently weighed, pooled and homogenized. The homogenates were kept in flasks at -20°C until analyzed. The digestive gland index (DGI) was calculated a s : F. wet wt digestive gland -1 DGI =/(wet wt shrimp 1 × 100. L - wet wt reproductive system)_] This modified index avoids a misleading decrease in the digestive gland index caused merely by increasing the reproductive system size (Clarke, 1977). Text data refers to the mean digestive gland index defined as the average value of digestive gland indices in each sex, stage of gonad maturation and season. The statistical treatment of morphometric data was conducted by an analysis of variance (ANOVA) at a level of significance of 1% or 5%. The values of the
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WALTERHORACIOJECKEL et al.
Table 1. Seasonalbody weightand digestiveglandweightrelationshipsin femalesof the shrimpPleotlcus muelleri, San Jorge Gulf, 1984-1985 Sampling Ovary Average body Average digestive Linear correlation date stage N weight (g) gland weight 0g) coefficient DGI + SD Winter Si 30 60.1 (46.7-73.4) 4.26 (2.70-5.42) 0.80 7.37 + 0.76 (6 Aug. 1984) Spring S1 17 34.3 (14.0-54.5) 2.32 (0.58-3.74) 0.92 6.58 _ 1.16 (l Nov, 1984) Summer S2 13 51.5 (34.5--68.5) 3.21 (1.95-5.83) 0.84 5.99 + 1.18 (15 Jan. 1985) $3 21 45.6 (32.0-59.2) 2.38 (0.91-4.22) 0.93 5.38 + 1.11 Autumn St 19 32:.3 (26.0-38.5) 2.20 (1.93-2.89) 0.54 6.93 +_.0.71 (1 Apr. 1985) S~, immatureovary; $2, incipientmaturation; S3, completematurity; N, number of individuals;DGI + SD, digestivegland index+ standard deviation. Extreme values in parentheses. mean digestive gland index were compared by means of the Tukey's paired contrast (Box et al., 1978). The term "level" designates the concentration of a biochemical component (in percentage or in mg/g of wet weight). The absolute amounts were calculated for a standard female of 35 g and a standard male of 28 g, on the basis of the mean values of the digestive gland index (and the reproductive system) and the corresponding level of a given biochemical component. RE,SULTS M o r p h o m e t r i c analysis
Table 1 summarizes the relationship between the digestive gland weight and the total body weight of females throughout the year. In females of winter, spring and summer the digestive gland weight was positively correlated (P < 0.05) with the total body weight whereas in the a u t u m n there was no correlation. Although the mean D G I had higher values in immature females (St) than in maturing females ($2) and those fully mature ($3), the study of paired contrast showed no significant differences (P < 0.05) between the values belonging to: (1) females St of the different seasons; (2) females St of spring and $2 of summer; and (3) females $2 and $3. Table 2 summarizes the relationship between the digestive gland weight and the total body weight of males throughout the year. In males ($3) of winter and summer, and in those St of spring, the digestive gland weight was positively correlated (P < 0.05) with the total body weight whereas in males ($3) of spring and a u t u m n there was no correlation. The study of paired contrast showed that the mean D G I was significantly (P < 0.01) lower in summer than in the other seasons. Biochemical composition
Table 3 shows the variation in the biochemical composition of the digestive gland of females
throughout the year. Water level in immature females (S~) varied inversely to that of lipid s. The latter increased notably from winter to spring and reached their maximum value in autumn. The ratio neutral lipids/polar lipids (NL/PL) increased in the same sense from 1.73 in winter to 13.48 in autumn, due to an accumulation of neutral lipids and a decrease of polar lipids. Protein concentration in females St did not vary significantly between the different seasons. Females $2 and $3 had lower levels of total lipids than females S~. In maturing females ($2) both, polar and neutral lipids fell markedly whilst water and protein increased. During the period of maturation from $2 to $3 there was no significant variation in lipid level, although an increase of protein at the expense of water was observed. Ash varied within a narrow range throughout the year. Total carbohydrate level (determined by the difference to 100 of the summation of the remainder of the biochemical components) represented ca 3-7% of wet weight and varied with no clear seasonal pattern. Table 4 shows the variation in the biochemical composition of the digestive gland of males throughout the year. The level of total lipids in mature males ($3) gradually increased from winter toward the autumn; however, the ratio NL/PL showed marked seasonal changes. F r o m winter to summer the level of polar lipids increased and that of neutral lipids decreased, whereas the inverse occurred from summer to autumn. Consequently, the ratio N L / P L varied from 5.94 in winter to 0.97 in summer and to 13.76 in autumn. Immature males (St) had higher levels of neutral and polar lipids than those mature of spring. Water level varied inversely to that of neutral lipids in males $3 whereas the concentrations of protein and ash did not vary significantly with the seasons nor with the grade of testes development. Total carbohydrate represented ca 3.5-10.0% of the wet weight and varied with no clear seasonal pattern.
Table 2. Seasonalbody weightand digestivegland weight relationshipsin malesof the shrimp Pleoticus muelleri, San Jorge Gulf, 1984--1985 Sampling MRS Average b o d y Averagedigestive Linear correlation date stage N weight (g) gland weight (g) coefficient DGI _ SD Winter S3 21 37.1 (27.5-45.0) 1.88 (0.74--2.87) 0.89 5.20 + 1.24 (6 Aug. 1984) Spring S~ 16 23.8 (14.0-35.0) 1.21 (0.38-1.85) 0.89 5.32 _ 0.60 (1 Nov. 1984) S3 11 24.2 (19.0-30.0) 1.19 (0.87-1.57) 0.68 5.08+0.64 Summer S3 33 28.0 (20.5-40.0) 1.09 (0.72-2.19) 0.80 3.99 + 0.62 (15 Jan. 1985) Autumn S3 20 23.3 (17.3-28.0) 1.28 (0.78-1.86) 0.54 5.69 _ 0.95 (1 Apr. 1985) MRS, male reproductivesystem; S~, immature reproductivesystem; $3, mature reproductivesystem. Other details as in Table 1.
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Seasonal composition o f shrimp--digestive gland Table 3. Seasonal variation in the percentage (of wet wt) biochemical composition of the digestive gland in females of the shrimp Pleoticus muelleri, San Jorge Gulf, 1984-1985 Ovary stage
Water
TL
Lipids NL
PL
Crude protein
Ash
Summer
S~ S1 S: $3
55.9 50.6 66.0 61.5
24.0 29.4 15.5 15.3
15.2 23.1 12.4 12.8
8.8 6.3 3.1 2.5
11.4 1t. 1 13.5 16.8
1.7 1.6 2.0 2.0
Autumn
S~
49.4
33.3
31.0
2.3
10.9
1.9
Season Winter Spring
S~, immature ovary; $2, incipient maturation; S3, complete maturity. TL, total lipids; NL, neutral lipids; PL, polar lipids.
Lipid classes Table 5 shows the variation of the neutral lipid composition in the digestive gland of females. Free fatty acids and triacylglycerols were the major lipid classes followed by alkyldiacylglycerols and the low R f components (partial glycerols, free sterols and minor amounts of pigments). The concentration of the different neutral lipid classes in females St varied during the year. Free fatty acids were remarkably low in winter and increased toward the autumn; alkyldiacylglycerols showed a similar trend. Triacylglycerol concentration was relatively high in winter and spring, and reached the maximum in autumn. The low Rf components had similar values in winter and autumn with a peak in spring. At early oogenesis ($2) all neutral lipid classes, except alkyldiacylglycerols, fell markedly. During the period of maturation from S: to $3, free fatty acids and triacylglycerols decreased slightly whilst the low Rf components increased and alkyldiacylglycerols remained fairly constant. Table 6 shows the variation of the neutral lipid composition in the digestive gland of males. Free fatty acids and triacylglycernls were the major lipid classes, followed by alkyldiacylglycerols and the low Rr components. The concentration of the different neutral lipid classes in mature males ($3) varied during the year. Free fatty acids were remarkably low in winter and high in autumn. Triacylglycerol concentration had important fluctuations, with a maximum value in winter and a minimum in spring whereas alkyldiacylglycerols were high in autumn. With the maturation of testes (from St to $3) in spring, free fatty acids and triacylglycerols markedly decreased whilst alkyldiacylglycerols decreased slightly and the low P~ components increased. Fatty acids Table 7 shows the variation of the fatty acid composition in the digestive gland of females
throughout the year. Predominant fatty acids were 16:0, 1 6 : 1 n - 7 , 1 8 : 1 n - 9 , 20:1, 2 0 : 5 n - 3 and 22: 6n - 3. The neutral lipids of females St of winter and spring had higher levels of saturated and monoenoic fatty acids than females $2 and $3 of summer and St of the autumn. It should be pointed out that fatty acids 16:0, 16:1 and 18:1 had a similar pattern throughout the year, and they were responsible for the variations in the levels of saturated and monoenoic fatty adds. The acid 20:1 reached the maximum value in summer with no significant difference of level between females $2 and $3. The ratio 22: 6/20:5 had a minimum value of 0.68 in spring and a maximum of 1.36 in autumn. The fatty acid composition of polar lipids, contrary to neutral lipids, showed no clear seasonal variation. The level of acid 16:0 in particular, was higher in the mature females than in immature ones. Polar lipids of immature females of spring and autumn were richer in polyenoic fatty acids than those of immature females of winter and the mature ones. Table 8 shows the variation of the fatty acid composition in the digestive gland of males throughout the year. Predominant fatty acids were 16:0, 16: In - 7, 18:1n - 9 , 20:5n - 3 and 22:6n - 3. The general fatty acid composition had scarce variations during the year. In summer, polar lipids were rich in polyenoic fatty adds, especially 2 0 : 5 n - 3 and 22:6n - 3, at the expense of the acids 16:0, 16:1 and 18:1 whereas in autumn the level of acid 16:0 increased markedly and polyenoic fatty acids decreased. The ratio 22:6/20:5 in neutral lipids varied between 0.64 in spring and 1.32 in autumn. DISCUSSION
Females The morphometric analysis of female shrimp showed that the digestive gland weight is linearly correlated to the total body weight during the major part of the year. The lack of correlation in autumn is not due to the grade of ovary development (Table 1)
Table 4. Seasonal variation in the percentage (of wet wt) biochemical composition of the digestive gland in males of the shrimp Pleoticus mueUeri, San Jorge Gulf, 1984-1985 MRS stage
Water
TL
Lipids NL
PL
Crude protein
Ash
Winter
S3 $1
52.3 51.3
24.3 31.0
20.8 18.3
3.5 12.7
11.8 11.6
1.6 1.6
Spring
S3
54.0
26.2
14.6
11.6
12.0
1.7
Summer
S3
53.9
29.0
14.3
14.7
11.9
1.8
Autumn
S3
50.6
31.0
28.9
2.1
11.3
2.1
Season
MRS, male reproductive system; $1, immature reproductive system; $3, mature reproductive system; TL, total lipids; NL, neutral lipids; PL, polar lipids.
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WALTERHORACIOJECKELet al. Table 5. Seasonal variation in the concentration (mg/g of wet wt) of neutral lipid classes in the digestive gland of female shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 Ovary stage Low Rf components Free fatty acids Triacylglycerols Alkyldiacylglycerols
Winter S1
Spring SI
S2
Summer S3
Autumn SI
41.2 23.2 65.6 22.0
54.6 84.0 65.4 26.2
15.2 50.0 32.6 26.1
30.1 43.3 28.3 26.4
38.7 122.7 109.8 38.7
Sl, immature ovary; $2, incipient maturation; $3, complete maturity. Low Rf components: partial glycerols, free sterols, pigments.
nor to the molting stage, since a previous work showed a high correlation between the digestive gland weight and the total body weight from stage B to D t of the molting cycle in immature females (Jeckel et al., 1990). On the other hand, it is interesting to note that in the mature males of autumn the digestive gland weight was neither correlated to the total body weight (Table 2). Thus, the lack of correlation appears to represent a characteristic of the population sampled. In many marine invertebrates the transference of nutrients from somatic storage organs to the ovary determines a decrease in size of the former during gonad development (Lawrence, 1976). P. muelleri increases notably the ovary index during oogenesis, from 1.75 + 0.60 in females S t of spring to 3.51 _ 1.17 in females $2 and to 7.98 + 2.01 in those fully mature ($3) of summer (Jeckel et al., 1989a). However, the statistical results of the present study show that only the D G I of females $3 differs significantly from those of females St throughout the year. This indicates that the decrease in relative size of the digestive gland is not concomitant with the increase of ovary development, and besides, the possible transference or consumption of nutrients only influences slightly on the digestive gland size at the completion of oogenesis. Lipid and protein are the major biochemical components of the digestive gland throughout the year. Lipid content in immature females (St) is low in winter and increases in spring reaching the maximum in autumn. This variation is characterized by the accumulation of neutral lipids, particularly free fatty acids and triacylglycerols, and probably reflects the annual cycle of feeding. The remarkably high ratio NL/PL in autumn was also observed in the digestive gland of males (Table 4) and the reproductive tissues (Jeckel et al., 1989a, b), and muscle of both sexes {Jeckel et al., 1991). This suggests that the shrimp would notably intensify feeding in autumn, after the peak of reproductive activity in summer (see below) and all of its tissues accumulate reserves, a part of which are probably used to undergo the winter. The fall of free fatty acids of the digestive gland in winter suggests that these components are consumed prior to triacylglycerols during periods of lower food
quality and/or availability (Table 5). The protein content in immature females, contrary to lipids, does not vary significantly during the year. The most striking results found in the females were the marked fall of all lipid components (excepting alkyldiacylglycerols) in the digestive gland of females $2 and $3, as well as the increase of protein concentration. A study on the biochemical composition of the stomachal content of P. muelleri collected from its natural habitat, showed that the shrimp does not interrupt feeding during the gonad maturation in summer (Jeckel et al., unpublished data). Thus, starvation as a cause of the lipid fall in females of summer can be discarded. On the other hand, the molting cycle of P. muelleri as in decapods in general, involves lipid consumption of the digestive gland from midpremolt to eedysis (see Jeckel et al., 1990). Maturing females, however, do not molt since growth and maturation are both energetically expensive and tend to be separated in time to avoid metabolic competition (Rice and Armitage, 1974; Adiyodi, 1985; Abell6, 1989). Besides, nearly 80% of females $3 used in this study had spermatophores attached to the thelycum (Jeckel et al., 1989a); these shrimps do not molt because it would imply the loss of spermatophores with the exuviae and consequently the failure of fertilization. Hence, the fall of lipids in the digestive gland of females $2-S 3 seems to be exclusively due to their utilization in the oogenesis process. The expression in absolute values can be suitable to describe the changes in biochemical composition of the digestive gland during oogenesis in summer. The calculations for a standard female (from Tables 1 and 3, and Jeckel et aL, 1989a) show that from stage Sm in spring to stage S2 in summer, the digestive gland loses 352 mg of lipid (with no selectivity in classes, Table 5) and accumulates 27.5 mg of protein whereas the ovary stores 27 mg of lipid and 129 mg of protein. Thus, the possible transference of lipids from the digestive gland to the ovary at early oogenesis would not explain the marked fall of lipids in the digestive gland of females $2. The fate of these lipids is not clear; perhaps they could be utilized at this stage for the synthesis in situ of ovary proteins. From early maturation ($2) until the complete ovary
Table 6. Seasonal variation in the concentration (mg/g of wet wt) of neutral lipid classes in the digestive gland of male shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 MRS stage
Winter S3
S1
Spring S3
Summer S3
Autumn S3
Low Rf components Free fatty acids Triacylglycerols Alkyldiacylglycerols
17.1 31.8 129.7 29.4
27.9 71.3 52.1 31.7
43.8 48.6 25.4 28.1
4.6 50.7 55.4 32.3
72.3 104.3 64.2 48.2
S~, immature reproductive system; S3, mature reproductive system. Low Rf components: partial glycerols, free sterols, pigments.
S e a s o n a l c o m p o s i t i o n o f shrimp---digestive g l a n d
257
Table 7. Relative per cent seasonal variation in the fatty acid composition of polar (PL) and neutral (NL) lipid fractions in the digestive gland of female shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 Winter SI PL NL Fatty acid 12:0 14:0 14:1 15:0 15:1 16:0 16:In - 7 16:2 + 17:0 16:3 18:0 18:ln - 9 18:2n - 6 18:3n - 6 18:3n - 3 20:1 (n - 9 + n - 11) 20:2n - 6 20:4n - 6 20:4n - 3 + 22:1 20:5n - 3 22:4n - 6 24:1 22:5n - 3 22: 6n - 3 Unidentified Saturated Monocnoie Polyenoic
Spring Sl PL NL
-2.9 1.7 2.1 2.9 19.8 13.2 -3.1 4.5 17.8 1.6 1.6 -4.0 2.1 3.6 -11.8 2.4 --4.8 --
1.0 3.2 1.5 1.7 1.7 17.5 15.2 1.6 2.6 4.1 20.1 0.9 1.2 -6.3 0.9 1.5 1.8 6.4 1.7 0.6 1.4 6.6 0.5
0.5 2.6 0.9 1.0 0.9 13.4 13.7 -1.9 2.4 17.4 1.4 1.4 0.7 7.2 1.4 2.1 1.8 14.0 1.0 0.8 1.8 11.7 --
0.8 3.3 1.0 1.3 0.9 16.8 13.3 1.5 2.0 4.4 20.7 1.4 1.5 -7.9 0.8 1.6 1.2 10.1 0.9 . 1.2 6.9 --
29.3 39.6 31.0
28.3 46.3 24.9
19.9 41.8 38.3
27.4 44.4 27.8
Summer PL
NL
PL
NL
Autumn $1 PL
-3.7 1.7 1.9 2.4 21.6 8.8 2.1 1.9 3.7 15.4 1.3 1.4 0.8 5.6 1.8 3.0 -9.2 2.0
-3.9 1.5 1.8 2.6 25.0 9.3 1.7 1.4 3.8 16.9 -0.5 -4.5 1.4 2.7 1.1 10.3 1.6
2.6 5.8 3.2
0.6 2.7 1.0 1.2 1.4 12.0 8.7 1.7 1.7 5.1 15.7 1.5 2.1 0.5 9.9 1.9 3.3 2.9 10.5 2.9 . 2.8 7.5 2.1
2.1 5.9 1.8
0.8 3.4 1.2 1.3 1.5 9.2 7.6 1.7 1.7 5.8 13.0 1.5 1.4 0.5 9.9 1.6 2.4 2.3 11.4 3.2 1.2 4.6 9.4 2.9
0.6 3.2 0.9 1.5 1.4 19.9 8.6 1.6 2.3 2.9 14.7 1.9 1.8 1.8 4.4 2.9 3.2 3.7 8.4 2.9 -1.3 8.6 1.4
0.7 3.4 0.9 1.2 0.8 16.5 10.7 1.2 1.7 3.2 18.3 1.8 1.4 1.3 6.3 1.3 1.7 3.3 8.8 1.0 -1.3 12.0 1.1
32.0 33.9 30.9
22.5 38.2 37.1
35.4 35.4 27.4
21.4 35.6 39.8
28.9 31.9 37.8
25.6 38.7 34.6
$2
.
.
$3
NL
S~, immature ovary; $2, incipient maturation; $3, complete maturity. - - , less than 0.5% or not detected.
development ($3) the digestive gland losses 37 mg of lipid and accumulates 33 mg of protein, whilst the ovary stores 120mg of lipid and 371 mg of protein. Thus, in this period the digestive gland contributes poorly with organic reserves to the developing ovary; consequently, the female shrimp would mainly
depend on its immediate food intake to satisfy the high vitellogenic requirements of triacylglycerols, phospholipids and protein (Jeckel et ai., 1989a). In the crab Carcinus maenas and the prawn Penaeus indicus the direct input of lipids from feeding were also preponderant (Heath and Barnes, 1970; Galois,
Table 8. Relative per cent seasonal variation in the fatty acid composition of polar (PL) and neutral (NL) lipid fractions in the digestive gland of male shrimp, Pleoticus muelleri, San Jorge Gulf, 1984--1985 Winter
Spring
S3
Summer
Si
S3
Autumn
S3
S3
PL
NL
PL
NL
PL
NL
PL
NL
PL
NL
Fatty acid 12:0 14:0 14:1 15:0 15:1 16:0 16:ln - 7 16:2 + 17:0 16:3 18:0 18: In - 9 18: 2n - 6 18:3n - 6 18:3n - 3 20:l(n - 9 + n - I1) 20: 2n - 6 20:4n - 6 20:4n - 3 + 22:1 20"5n - 3 22:4n - 6 24:1 22: 5n - 3 22:6n - 3 Unidentified
1.3 2.5 1.3 1.5 1.7 12.9 12.9 1.7 2.8 2.8 16.7 1.4 1.5 0.9 5.4 1.6 2.9 1.2 10.1 1.9 -1.9 11.4 1.6
1.3 3.1 1.4 1.7 2.0 14.4 12.9 1.8 3.0 4.3 18.7 1.2 1.6 1,0 7.2 1,3 2,3 1,6 7.0 1.5 -2.0 7.6 1.1
-3.6 1.1 1.3 1.1 13.6 12.1 1.3 2.0 2.9 16.4 1.6 1.4 0.7 7.2 1.6 2.0 2.4 13.5 0.7 -0.9 11.1 0.7
0.9 3.7 1.0 1.4 1.2 15.5 11.6 1.5 1.9 4.8 19.4 0.5 0.7 -7.7 0.7 1.9 1.9 11.7 1.0 0.6 1.3 8.7 --
0.8 3.8 !.0 1.2 1.0 13.6 12.2 1.3 2.3 3.2 16.8 1.3 1.5 0.7 7.0 1.4 2.1 1.5 13.8 1.5 0.9 2.0 9.1 --
-4.1 1.0 1.3 1.0 15.9 11.6 1.5 2.0 5.2 18.6 1.5 1.3 -8.5 1.3 1.9 -11.5 1.0 0.8 1.5 7.4 1.0
0.5 2.7 0.9 1.1 0.9 10.4 7.8 1.3 1.7 3.4 14.4 1.3 1.1 0.8 7.5 1.7 3.5 1.6 15.3 2.5 0.7 3.2 14.7 1.0
1.1 3.5 1.1 1.4 l.l 16.1 8.5 1.7 1.5 6.3 19.8 1.4 1.3 0.6 8.5 1.6 2.0 1.8 7.7 1.4 -1.8 5.3 4.5
-1.3 1.7 1.7 1.3 22.2 8.8 1.3 1.9 3.0 17.6 0.6 -0.9 5.0 1.0 1.9 3.0 9.9 ---11.8 1.4
-3.9 0.9 1.3 0.8 16.9 10.3 1.2 1.8 3.9 19.4 1.4 1.3 1.2 6.4 1.2 1.6 3.0 9.4 0.5 0.5 0.6 12.4 --
Saturated Monoenoic I: Polyenoic
21.9 38.6 37.9
25.7 43.0 30.2
22.1 39.4 37.4
27.1 42.5 30.2
23.2 39.6 37.2
27.3 41.5 30.2
18.8 33.0 47.3
29.3 39.9 26.4
28.9 35.9 30.2
26.6 39.8 33.5
S~, immature reproductive system; $3, mature reproductive system. - - , less than 0.5% or not detected.
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WALTERHORACIOJECKELet al.
1984). In other decapods, however, the contribution in organic reserves of the digestive gland to the developing ovary was more important (Allen, 1972; Clarke, 1977; Kulkarni and Nagabhushanam, 1979; Teshima and Kanazawa, 1983). Protein storage in the digestive gland during oogenesis could be due to a decrease in the proteolytic activity of this organ in order to maintain an alternative source of energy. The scarce lipid supply from the digestive gland to the ovary, from $2 to $3, consisted of free fatty acids, triacylglycerols and phospholipids, whereas the increase of the low Rf components in the digestive gland was probably due to an accumulation of hydrolysis products (Table 5). P. muelleri appears to be lesser selective than the prawn Penaeus japonicus as the latter only transferred triacylglycerols from the digestive gland to the ovary during vitellogenesis (Teshima and Kanazawa, 1983). The fatty acid composition of the digestive gland is typical of marine decapods, with high levels of acids 16: 0, 16: In - 7, 18 : In - 9 and a marked predominance of the n - 3 polyenoic acids, mainly 20: 5 and 22:6 (Addison et al., 1972; Gopakumar and Nair, 1975; Guary et al., 1975; Middleditch et al., 1980; Chapelle, 1986). Fatty acid 20:1 reaches higher levels in the digestive gland than in the remainder of shrimp tissues (Jeckel et al., 1989a, b, 1991) as appears to be a common feature in several decapods (Allen, 1971; Teshima and Kanazawa, 1983). The fatty acid composition varied in both polar and neutral lipids of the digestive gland throughout the year. However, total fatty acid composition should reflect that of neutral lipids as this fraction is quantitatively more important (Tables 3 and 7). The most notable changes in the fatty acid composition of the digestive gland occur in summer during the ovary development. Saturated and monoenoic fatty acids of neutral lipids decrease with the progress of oogenesis, due to the apparent selective utilization of acids 16:0, 16:1 and 18:1; consequently, the level of polyenoic acids increases. Jeckel et al. (1989a) found a significant increase in the level of palmitic acid (16:0) in the ovary of P. muelleri during vitellogenesis, but not so of acids 16:1 and 18:1. Although palmitic acid is efficiently synthesized de novo by crustaceans (Castell, 1981), it cannot be ignored that at least in part this acid is transferred from the digestive gland to the developing ovary, whereas the acids 16:1 and 18:1 could be consumed as a source of energy. Teshima and Kanazawa (1983) found no significant variations in the fatty acid composition of the digestive gland and ovary during vitellogenesis of P. japonicus, thus suggesting that the fatty acid metabolism during this process varies considerably from one species to another. It is interesting to note that the ratio 22: 6/20: 5 in neutral lipids of the digestive gland and ovary varies with a similar pattern throughout the year. The origin of these fatty acids in the shrimp and the possible causes of their variations in relation to food quality have been discussed in previous studies (Jeckel et aL, 1989a, b). The variation in the fatty acid composition of polar lipids showed no clear seasonal trends. The major
part of these lipids would not be biomembrane components of the digestive gland but possibly reserves, judged by the marked variations in their contents (Table 3) and fatty acid levels. These variations could be due to diverse factors acting simultaneously, such as the dietary input, turnover, and the lipid transport mechanisms of the shrimp. Males
The relationship between the digestive gland weight and the total body weight is more variable in males than in females, since in males $3 of spring and autumn no correlation was found (Table 2). Although the relative size of the digestive gland of males as well as in females decreases in summer, the concomitant changes in the biochemical composition of this organ differ remarkably between sexes as is discussed below. The amount of lipid in males $3 increases from winter toward the autumn (except in summer, see below) which reflects the seasonal dietary changes discussed in females (Tables 3 and 4). However, the ratio NL/PL in males, as opposed to females, decreases from winter to summer with a striking increase of polar lipids. This suggests that the lipid metabolism of the digestive gland differs considerably between sexes during the year. Phospholipids are generally recognized because of their roles in transport processes and in the structural and functional maintenance of biomembranes (Chapelle, 1986). However, the remarkable variation in phospholipid content in the digestive gland of males suggests again a role as reserves in P. muelleri. Phosphoglycerides as depot lipids have also been proposed in adult krill Euphausia superba (Saether et al., 1985) and fish larvae (Tocher et al., 1985). In winter, the neutral lipids of males $3 (similarly to females) are rich in triacylglycerols, with the suggestion that the digestive ~ gland would utilize mainly free fatty acids during periods of low food availability (Table 6). In spring, the lipid composition of immature males (SI) differs markedly from that of mature males ($3). The fall of lipids in the digestive gland from Sl to S3 appears to be exclusively due to their utilization in the spermatogenesis process. The calculations for a standard male (from Tables 2 and 4) show that from stage S1 to S3 the digestive gland losses 65 mg of neutral lipids and 24 mg of polar lipids, whilst the protein content does not vary significantly. The lipid content of the reproductive system (testes plus spermatophores) does not vary appreciably with maturation whereas total nitrogen content increases from 29.4 to 35.3 mg (Jeckel et al., 1989b). This would suggest that nitrogenous compounds of the reproductive system are synthesized in situ during spermatogenesis, at least in part at the expense of the digestive gland. Free fatty acids and triacylglycerols (Table 6) would be utilized as a source of energy whereas phospholipids could be involved in transport processes. The results expressed in terms of concentration may lead to erroneous conclusions when values of an organ changing its size are compared. This may occur with the shrimp in summer, when the percentage values of biochemical components are high but the digestive gland size is around one-fifth smaller than
Seasonal composition of shrimp--digestive gland in the other seasons. In absolute amounts a standard male in summer (DGI = 3.99) would contain 108 mg of lipid and 44 mg of protein less than a hypothetic male (in summer) with a DGI = 5.32 (average index of males $3 of winter, spring and autumn). These morphometric and biochemical changes in the digestive gland of male shrimp in summer could indicate symptoms of starvation. It has been observed that starved marine invertebrates have the capacity to resorb body tissues and thus utilize biochemical components without affecting their levels (see Lawrence, 1976). It is worth mentioning a preliminary survey during a breeding season that showed stomachal content in male shrimp had 50% lesser lipid than that of females, thus suggesting energetically lower food in the formers in this period (Jeckel et al., unpublished data). The results of the present study would agree with those observations and lead us to postulate that males may undergo a metabolic stress in summer with the consequent degradation of tissues in the digestive gland. It is interesting to note that in spite of the morphometric changes, the absolute amount of polar lipid remains fairly constant between spring (DGI = 5.08) and summer (DGI = 3.99). This would suggest that the shrimp tends to conserve phospholipids in the digestive gland. The glycerophospholipid content could be maintained at the expense of partial glycerols (Table 6). Interestingly, Heath and Barnes (1970) noted the rise in phospholipid possibly at the expense of neutral fat in starved Carcinus maenas, but they could not explain the causes of such increase. In the case of the shrimp, phospholipids would appear to be reserved for some specific process probably occurring after the breeding period. Neutral lipid consumption may be accompanied by cell destruction with the consequent metabolization of protein, although utilization of non-protein nitrogenous compounds cannot be totally discarded. Possibly, males change their feeding habits, eat little, and increase locomotory activity during mating in summer; consequently, their energetic requirements would exceed the dietary input, as occurs in other species (Armitage et al., 1972; Tsai et al., 1984). It is concluded that, although the relative size of the digestive gland decreases in mature shrimps ($3) of summer, the causes of this morphometric variation as well as the changes in biochemical composition, differ remarkably between sexes. In autumn, the ratio NL/PL reaches the maximum value and is very similar in both sexes (Tables 3 and 4). Luxuriant feeding in this season appears to be the preponderant factor determining the lipid composition of the digestive gland, characterized by the accumulation of neutral lipids (Tables 5 and 6). The fatty acid spectrum in the digestive gland of both sexes, as well as the similar pattern in the ratio 22: 6/20:5 of neutral lipids, suggest an important influence of diet throughout the year. However, males had scarce variations in the level of major fatty acids, except in polar lipids in summer (Table 8). In this season, the increase in level of polyenoic fatty acids is apparently due to a selective utilization of the acids 16: 0, 16:1 and 18 : 1. Thus, the decrease in size of the digestive gland would not involve the loss of polar lipids (Tables 2 and 4) but a qualitative change in this fraction. With the fall of polar lipids in autumn the
259
digestive gland losses substantial amounts of polyenoic fatty acids, which are apparently replaced by palmitic acid (16:0). Such variation of fatty acids is not observed in females, thus suggesting that the causes would not be of a dietary origin and, at least in part, palmitic acid could be synthesized de novo by males. It has been observed for crustaceans and fish that chain elongation and desaturation of fatty acids tend to increase as a response to low environmental temperatures (Castell, 1981). The water temperature in the natural habitat of the shrimp (at a depth of 36-86 m) ranges from 6.7°C in winter (August) to 13.9°C in summer (January) (Jeckel et aL, 1989a). Consequently, an increase of polyenoic fatty acids could be expected to occur in winter, but this is not found to be the case. Probably, the fatty acids of dietary origin and their accumulation or selective consumption are preponderant in determining the lipid composition in the digestive gland of the shrimp. Acknowledgements--The authors are indebted to Lic. A. Ptrez for assistance in statistical calculations, and to Lic. S. De Marco who revised the translation of the manuscript. REFERENCES
Abell6 P. (1989) Reproduction and moulting in Liocarcinus depurator (Linnaeus, 1758) (Brachyura: Portunidae) in the Northwestern Mediterranean sea. Scient. Mar. 53, 127-134. Addison R. F., Ackman R. G. and Hingiey J. (1972) Lipid composition of the queen crab (Chionoecetes opilio). J. Fish. Res. Bd Can. 29, 407-411. Adiyodi R. G. (1985) Reproduction and its control. In The Biology of Crustacea (Edited by Bliss D. E. and Mantel L. H.), Vol. 9, pp. 147-215. Academic Press, New York. Allen W. V. (1971) Amino acid and fatty acid composition of tissues of the Dungeness crab (Cancer magister). J. Fish. Res. Bd Can. 28, 1191-1195. Allen W. V. (1972) Lipid transport in the Dungene3s crab, Cancer magister Dana. Comp. Physiol. Biochem. 43, 193-207. Armitage K. B., Buikema A. L., Jr and Willems N. J. (1972) Organic constituents in the annual cycle of the crayfish Orconectes nais (Faxon). Comp. Biochem. Physiol. 41, 825-842. Armitage K. B., Buikema A. L., Jr and Willems N. J. (1973) The effect of photoperiod on organic constituents and molting of the crayfish Orconectes nais (Faxon). Comp. Biochem. Physiol. 44, 431-456. Box G. E. P., Hunter W. G. and Hunter J. S. (1978) Statistics for Experimenters. An Introduction to Design, Data Analysis, and Model Building. John Wiley, New York. Castell J. D. (1981) Fatty acid metabolism in crustaceans. Proceedings of the Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Special Publication No.
2, October. Chapelle S. (1986) Aspects of phospholipid metabolism in crustaceans as related to changes in environmental temperatures and salinities. Comp. Biochem. Physiol. 84B, 423-439. Clarke A. (1977) Seasonal variations in the total lipid content of Chorismus antarcticus (Pfeffer) (Crustacea: Decapoda) at South Georgia. J. exp. mar. Biol. Ecol. 27, 93-106. Dall W. (1981) Lipid absorption and utilization in the Norwegian lobster, Nephrops norvegicus (L.). J. exp. mar. Biol. Ecol. 50, 33-45.
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Galois R. G. (1980) Le mttabolisme des lipides chez Penaeus japonieus Bate: teneur en eau et incorporation de lipides dam les tissus au cours du cycle d'intermue. Tethys 9, 371-377. Galois R. G. (1984) Variations de la composition lipidique tissnlaire au cours de la vitellogenese chez la crevette Penaeus indicus Milne Edwards. J. exp. mar. Biol. Ecol. 84, 155-166. Gopakumar K. and Rajendranathan Nair M. (1975) Lipid composition of five species of Indian prawns. J. Sci. Fd Agric. 26, 319-325. Guary J. C., Kayama M. and Murakami Y. (1974) Lipid class distribution and fatty acid composition of prawn, Penaeus japonicus Bate. Bull. Jap. Soc. scient. Fish. 40, 1027-1032. Guary J. C., Kayama M. and Murakami Y. (1975) Variations saisonnieres de la composition en acides gras chez Penaeus japonicus (Crustacea: Deacapoda). Mar. Biol. 29, 335-341. Heath J. R. and Barnes H. (1970) Some changes in biochemical composition with season and during the moulting cycle of the common shore crab, Carcinus maenas (L.). J. exp. mar. Biol. Ecol. 5, 199-233. Hug,gins A. K. and Munday K. A. (1968) Crustacean metabolism. In Advances in Comparative Physiology and Biochemistry (Edited by Lowenstein O.), Vol. 3, pp. 271-378. Academic Press, New York. Jeckel W. H., de Moreno J. E. A. and Moreno V. J. (1989a) Biochemical composition, lipid classes and fatty acids in the ovary of the shrimp Pleoticus muelleri Bate. Comp. Biochem. Physiol. 92B, 271-276. Jeckel W. H., de Moreno J. E. A. and Moreno V. J. (1989b) Biochemical composition, lipid classes and fatty acids in the male reproductive system of the shrimp Pleoticus muelleri Bate. Comp. Biochem. Physiol. 9311, 807-811. Jeckel W. H., de Moreno J. E. A. and Moreno V. J. (1990) Changes in biochemical composition and lipids of the digestive gland in females of the shrimp Pleoticus muelleri (Bate) during the molting cycle. Comp. Biochem. Physiol. 96B, 521-525.
Jeckel W. H., de Moreno J. E. A. and Moreno V. J. (1991) Seasonal variations in the biochemical composition and lipids of muscle and carapace in the shrimp Pleoticus muelleri Bate. Comp. Biochem. Physiol. 98B, 261-266. Kulkarni G. K. and Nagabhushanam R. (1979) Mobilization of organic reserves during ovarian development in a marine penaeid prawn, Parapenaeopsis hardwickii (Miers) (Crustacea, Decapoda, Penaeidae). Aquaculture 18, 373-377. Lawrence J. M. (1976) Patterns of lipid storage in postmetamorphic marine invertebrates. Am. Zool. 16, 747-762. Middleditch B. S., Missler S. R., Hines H. B., McVey J. P., Brown A., Ward D. G. and Lawrence A. L. (1980) Metabolic profiles of penaeid shrimp: dietary lipids and ovarian maturation. J. Chromat. 195, 359-369. O'Connor J. D. and Gilbert L. I. (1969) Alterations in lipid metabolism associated with premolt activity in a land crab and a freshwater crayfish. Comp. Biochem. Physiol. 29, 889-904. Rice P. R. and Armitage K. B. (1974) The influence of photoperiod on processes associated with molting and reproduction in the crayfish Orconectes nais (Faxon). Comp. Biochem. Physiol. 47, 243-259. Saether O., Ellingsen T. E. and Mohr V. (1985) The distribution of lipid in the tissues of Antarctic krill Euphausia superba. Comp. Biochem. Physiol. 81B, 609-614. Teshima S. and Kanazawa A. (1983) Variation in lipid compositions during the ovarian maturation of the prawn. Bull. Jap. Soc. scient. Fish. 49, 957-962. Tocher D. R., Fraser A. J., Sargent J. R. and Gamble J. C. (1985) Lipid class composition during embryonic and early larval development in Atlantic herring (Clupea harengus, L.). Lipids 20, 84-89. Travis D. F. (1955) The molting cycle of the spiny lobster, Panulirus argus Latreille--II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. 108, 88-112. Tsai D. E., Chen H. and Tsai C. (1984) Total lipid and cholesterol content in the blue crab, Callinectes sapidus Rathbun. Comp. Biochem. Physiol. 78B, 27-31.
0305-0491/91 $3.00+ 0.00 © 1991PergamonPress pie
SEASONAL VARIATIONS IN THE BIOCHEMICAL COMPOSITION A N D LIPIDS OF THE DIGESTIVE GLAND IN THE SHRIMP PLEOTICUS MUELLERI BATE WALTERHORAClOJECKEL, JULIAELENAAIZPUNDE MORENOand VfCTORJORGE MORENO Instituto Nacional de Investigaci6n y Desarrollo Pesquero (INIDEP), C.C. 175 Playa Grande s/No. 7600, Mar del Plata, Argentina (Received 9 July 1990) Aimtract--l. Seasonal variations in the biochemical composition, lipid classes and fatty acids in the digestive gland of the shrimp Pleoticus muelleri from San Jorge Gulf, Argentina, were studied during 1984-1985. 2. The relative size of the digestive gland decreased significantlyin fully mature shrimps of both sexes during the summer. 3. Immature females and mature males gradually increased the level of total lipids from winter toward the autumn as a reflex of the annual cycle of feeding. 4. Total lipids fell markedly during the ovary maturation in summer whilst protein increased. Free fatty acids, triacylglycerolsand phospholipids were apparently transferred to the developing ovary; however, the immediate dietary supply of lipids and protein appeared to be preponderant from incipient maturation until the completion of ovary development. 5. Total lipids decreased with the maturation of the male reproductive system in spring with the suggestion of their utilization during spermatogenesis. 6. Maturing females appeared to use selectivelyfatty acids 16:0, 16:1 and 18:1 of neutral lipids during oogenesis in summer whereas males used the mentioned acids of polar lipids apparently due to metabolic stress. 7. The results are discussedin terms of concentration and absolute amounts in relation to physiological and ecological factors.
INTRODUCTION
MATERIALSAND METHODS
The digestive gland is generally regarded as a major storage organ in decapod crustaceans (Huggins and Munday, 1968; Allen, 1971; Guary et al., 1974; Teshima and Kanazawa, 1983). The study of the digestive gland is of considerable interest because of its roles in the accumulation and cyclic mobilization of reserves during the molting process (Travis, 1955; O'Connor and Gilbert, 1969; Galois, 1980; Jeckel et aL, 1990); the contribution with nutrients to the ovary during vitellogenesis (Armitage et al., 1973; Kulkarni and Nagabhushanam, 1979; Adiy0di, 1985); the mobilization of reserves during starvation (Heath and Barnes, 1970; Armitage et al., 1972) as well as in the digestion and absorption processes (Dall, 1981). The purpose of the present study was to characterize the seasonal pattern of the biochemical composition, lipid classes and fatty acids in the digestive gland of the marine shrimp Pleoticus muelleri, in relation to physiological factors (such as sex and stage of gonad maturation) and ecological factors (e.g. food quality and availability, and temperature). This paper complements previous works which reported the morphometric and biochemical changes in the reproductive system of the shrimp (Jeckel et al., 1989a, b). Hence, a great part of the results presented here are discussed in terms of concentration and absolute amounts in relation to the mentioned studies.
The organisms and the capture area (San Jorge Gulf, Argentina) as well as the biological and biochemical methods utilized in this study have been described in previous works (Jeekel et al., 1989a, b). The animals were separated according to the sex and stage of gonad maturation, and subsequently weighed. Because we were interested in general trends of each sex in the population studied, individual molting stages were ignored; however, shrimps with signs of imminent molt or recently molted were not used. In both sexes, Sl represents sexual immaturity; $2, incipient maturation; and $3, complete maturity. The male reproductive system referred to here comprised the set testes plus spermatophores. Digestive glands corresponding to each sex and stage of gonad maturation were carefully extracted from the shrimps inside an icebath. They were subsequently weighed, pooled and homogenized. The homogenates were kept in flasks at -20°C until analyzed. The digestive gland index (DGI) was calculated a s : F. wet wt digestive gland -1 DGI =/(wet wt shrimp 1 × 100. L - wet wt reproductive system)_] This modified index avoids a misleading decrease in the digestive gland index caused merely by increasing the reproductive system size (Clarke, 1977). Text data refers to the mean digestive gland index defined as the average value of digestive gland indices in each sex, stage of gonad maturation and season. The statistical treatment of morphometric data was conducted by an analysis of variance (ANOVA) at a level of significance of 1% or 5%. The values of the
253
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WALTERHORACIOJECKEL et al.
Table 1. Seasonalbody weightand digestiveglandweightrelationshipsin femalesof the shrimpPleotlcus muelleri, San Jorge Gulf, 1984-1985 Sampling Ovary Average body Average digestive Linear correlation date stage N weight (g) gland weight 0g) coefficient DGI + SD Winter Si 30 60.1 (46.7-73.4) 4.26 (2.70-5.42) 0.80 7.37 + 0.76 (6 Aug. 1984) Spring S1 17 34.3 (14.0-54.5) 2.32 (0.58-3.74) 0.92 6.58 _ 1.16 (l Nov, 1984) Summer S2 13 51.5 (34.5--68.5) 3.21 (1.95-5.83) 0.84 5.99 + 1.18 (15 Jan. 1985) $3 21 45.6 (32.0-59.2) 2.38 (0.91-4.22) 0.93 5.38 + 1.11 Autumn St 19 32:.3 (26.0-38.5) 2.20 (1.93-2.89) 0.54 6.93 +_.0.71 (1 Apr. 1985) S~, immatureovary; $2, incipientmaturation; S3, completematurity; N, number of individuals;DGI + SD, digestivegland index+ standard deviation. Extreme values in parentheses. mean digestive gland index were compared by means of the Tukey's paired contrast (Box et al., 1978). The term "level" designates the concentration of a biochemical component (in percentage or in mg/g of wet weight). The absolute amounts were calculated for a standard female of 35 g and a standard male of 28 g, on the basis of the mean values of the digestive gland index (and the reproductive system) and the corresponding level of a given biochemical component. RE,SULTS M o r p h o m e t r i c analysis
Table 1 summarizes the relationship between the digestive gland weight and the total body weight of females throughout the year. In females of winter, spring and summer the digestive gland weight was positively correlated (P < 0.05) with the total body weight whereas in the a u t u m n there was no correlation. Although the mean D G I had higher values in immature females (St) than in maturing females ($2) and those fully mature ($3), the study of paired contrast showed no significant differences (P < 0.05) between the values belonging to: (1) females St of the different seasons; (2) females St of spring and $2 of summer; and (3) females $2 and $3. Table 2 summarizes the relationship between the digestive gland weight and the total body weight of males throughout the year. In males ($3) of winter and summer, and in those St of spring, the digestive gland weight was positively correlated (P < 0.05) with the total body weight whereas in males ($3) of spring and a u t u m n there was no correlation. The study of paired contrast showed that the mean D G I was significantly (P < 0.01) lower in summer than in the other seasons. Biochemical composition
Table 3 shows the variation in the biochemical composition of the digestive gland of females
throughout the year. Water level in immature females (S~) varied inversely to that of lipid s. The latter increased notably from winter to spring and reached their maximum value in autumn. The ratio neutral lipids/polar lipids (NL/PL) increased in the same sense from 1.73 in winter to 13.48 in autumn, due to an accumulation of neutral lipids and a decrease of polar lipids. Protein concentration in females St did not vary significantly between the different seasons. Females $2 and $3 had lower levels of total lipids than females S~. In maturing females ($2) both, polar and neutral lipids fell markedly whilst water and protein increased. During the period of maturation from $2 to $3 there was no significant variation in lipid level, although an increase of protein at the expense of water was observed. Ash varied within a narrow range throughout the year. Total carbohydrate level (determined by the difference to 100 of the summation of the remainder of the biochemical components) represented ca 3-7% of wet weight and varied with no clear seasonal pattern. Table 4 shows the variation in the biochemical composition of the digestive gland of males throughout the year. The level of total lipids in mature males ($3) gradually increased from winter toward the autumn; however, the ratio NL/PL showed marked seasonal changes. F r o m winter to summer the level of polar lipids increased and that of neutral lipids decreased, whereas the inverse occurred from summer to autumn. Consequently, the ratio N L / P L varied from 5.94 in winter to 0.97 in summer and to 13.76 in autumn. Immature males (St) had higher levels of neutral and polar lipids than those mature of spring. Water level varied inversely to that of neutral lipids in males $3 whereas the concentrations of protein and ash did not vary significantly with the seasons nor with the grade of testes development. Total carbohydrate represented ca 3.5-10.0% of the wet weight and varied with no clear seasonal pattern.
Table 2. Seasonalbody weightand digestivegland weight relationshipsin malesof the shrimp Pleoticus muelleri, San Jorge Gulf, 1984--1985 Sampling MRS Average b o d y Averagedigestive Linear correlation date stage N weight (g) gland weight (g) coefficient DGI _ SD Winter S3 21 37.1 (27.5-45.0) 1.88 (0.74--2.87) 0.89 5.20 + 1.24 (6 Aug. 1984) Spring S~ 16 23.8 (14.0-35.0) 1.21 (0.38-1.85) 0.89 5.32 _ 0.60 (1 Nov. 1984) S3 11 24.2 (19.0-30.0) 1.19 (0.87-1.57) 0.68 5.08+0.64 Summer S3 33 28.0 (20.5-40.0) 1.09 (0.72-2.19) 0.80 3.99 + 0.62 (15 Jan. 1985) Autumn S3 20 23.3 (17.3-28.0) 1.28 (0.78-1.86) 0.54 5.69 _ 0.95 (1 Apr. 1985) MRS, male reproductivesystem; S~, immature reproductivesystem; $3, mature reproductivesystem. Other details as in Table 1.
255
Seasonal composition o f shrimp--digestive gland Table 3. Seasonal variation in the percentage (of wet wt) biochemical composition of the digestive gland in females of the shrimp Pleoticus muelleri, San Jorge Gulf, 1984-1985 Ovary stage
Water
TL
Lipids NL
PL
Crude protein
Ash
Summer
S~ S1 S: $3
55.9 50.6 66.0 61.5
24.0 29.4 15.5 15.3
15.2 23.1 12.4 12.8
8.8 6.3 3.1 2.5
11.4 1t. 1 13.5 16.8
1.7 1.6 2.0 2.0
Autumn
S~
49.4
33.3
31.0
2.3
10.9
1.9
Season Winter Spring
S~, immature ovary; $2, incipient maturation; S3, complete maturity. TL, total lipids; NL, neutral lipids; PL, polar lipids.
Lipid classes Table 5 shows the variation of the neutral lipid composition in the digestive gland of females. Free fatty acids and triacylglycerols were the major lipid classes followed by alkyldiacylglycerols and the low R f components (partial glycerols, free sterols and minor amounts of pigments). The concentration of the different neutral lipid classes in females St varied during the year. Free fatty acids were remarkably low in winter and increased toward the autumn; alkyldiacylglycerols showed a similar trend. Triacylglycerol concentration was relatively high in winter and spring, and reached the maximum in autumn. The low Rf components had similar values in winter and autumn with a peak in spring. At early oogenesis ($2) all neutral lipid classes, except alkyldiacylglycerols, fell markedly. During the period of maturation from S: to $3, free fatty acids and triacylglycerols decreased slightly whilst the low Rf components increased and alkyldiacylglycerols remained fairly constant. Table 6 shows the variation of the neutral lipid composition in the digestive gland of males. Free fatty acids and triacylglycernls were the major lipid classes, followed by alkyldiacylglycerols and the low Rr components. The concentration of the different neutral lipid classes in mature males ($3) varied during the year. Free fatty acids were remarkably low in winter and high in autumn. Triacylglycerol concentration had important fluctuations, with a maximum value in winter and a minimum in spring whereas alkyldiacylglycerols were high in autumn. With the maturation of testes (from St to $3) in spring, free fatty acids and triacylglycerols markedly decreased whilst alkyldiacylglycerols decreased slightly and the low P~ components increased. Fatty acids Table 7 shows the variation of the fatty acid composition in the digestive gland of females
throughout the year. Predominant fatty acids were 16:0, 1 6 : 1 n - 7 , 1 8 : 1 n - 9 , 20:1, 2 0 : 5 n - 3 and 22: 6n - 3. The neutral lipids of females St of winter and spring had higher levels of saturated and monoenoic fatty acids than females $2 and $3 of summer and St of the autumn. It should be pointed out that fatty acids 16:0, 16:1 and 18:1 had a similar pattern throughout the year, and they were responsible for the variations in the levels of saturated and monoenoic fatty adds. The acid 20:1 reached the maximum value in summer with no significant difference of level between females $2 and $3. The ratio 22: 6/20:5 had a minimum value of 0.68 in spring and a maximum of 1.36 in autumn. The fatty acid composition of polar lipids, contrary to neutral lipids, showed no clear seasonal variation. The level of acid 16:0 in particular, was higher in the mature females than in immature ones. Polar lipids of immature females of spring and autumn were richer in polyenoic fatty acids than those of immature females of winter and the mature ones. Table 8 shows the variation of the fatty acid composition in the digestive gland of males throughout the year. Predominant fatty acids were 16:0, 16: In - 7, 18:1n - 9 , 20:5n - 3 and 22:6n - 3. The general fatty acid composition had scarce variations during the year. In summer, polar lipids were rich in polyenoic fatty adds, especially 2 0 : 5 n - 3 and 22:6n - 3, at the expense of the acids 16:0, 16:1 and 18:1 whereas in autumn the level of acid 16:0 increased markedly and polyenoic fatty acids decreased. The ratio 22:6/20:5 in neutral lipids varied between 0.64 in spring and 1.32 in autumn. DISCUSSION
Females The morphometric analysis of female shrimp showed that the digestive gland weight is linearly correlated to the total body weight during the major part of the year. The lack of correlation in autumn is not due to the grade of ovary development (Table 1)
Table 4. Seasonal variation in the percentage (of wet wt) biochemical composition of the digestive gland in males of the shrimp Pleoticus mueUeri, San Jorge Gulf, 1984-1985 MRS stage
Water
TL
Lipids NL
PL
Crude protein
Ash
Winter
S3 $1
52.3 51.3
24.3 31.0
20.8 18.3
3.5 12.7
11.8 11.6
1.6 1.6
Spring
S3
54.0
26.2
14.6
11.6
12.0
1.7
Summer
S3
53.9
29.0
14.3
14.7
11.9
1.8
Autumn
S3
50.6
31.0
28.9
2.1
11.3
2.1
Season
MRS, male reproductive system; $1, immature reproductive system; $3, mature reproductive system; TL, total lipids; NL, neutral lipids; PL, polar lipids.
256
WALTERHORACIOJECKELet al. Table 5. Seasonal variation in the concentration (mg/g of wet wt) of neutral lipid classes in the digestive gland of female shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 Ovary stage Low Rf components Free fatty acids Triacylglycerols Alkyldiacylglycerols
Winter S1
Spring SI
S2
Summer S3
Autumn SI
41.2 23.2 65.6 22.0
54.6 84.0 65.4 26.2
15.2 50.0 32.6 26.1
30.1 43.3 28.3 26.4
38.7 122.7 109.8 38.7
Sl, immature ovary; $2, incipient maturation; $3, complete maturity. Low Rf components: partial glycerols, free sterols, pigments.
nor to the molting stage, since a previous work showed a high correlation between the digestive gland weight and the total body weight from stage B to D t of the molting cycle in immature females (Jeckel et al., 1990). On the other hand, it is interesting to note that in the mature males of autumn the digestive gland weight was neither correlated to the total body weight (Table 2). Thus, the lack of correlation appears to represent a characteristic of the population sampled. In many marine invertebrates the transference of nutrients from somatic storage organs to the ovary determines a decrease in size of the former during gonad development (Lawrence, 1976). P. muelleri increases notably the ovary index during oogenesis, from 1.75 + 0.60 in females S t of spring to 3.51 _ 1.17 in females $2 and to 7.98 + 2.01 in those fully mature ($3) of summer (Jeckel et al., 1989a). However, the statistical results of the present study show that only the D G I of females $3 differs significantly from those of females St throughout the year. This indicates that the decrease in relative size of the digestive gland is not concomitant with the increase of ovary development, and besides, the possible transference or consumption of nutrients only influences slightly on the digestive gland size at the completion of oogenesis. Lipid and protein are the major biochemical components of the digestive gland throughout the year. Lipid content in immature females (St) is low in winter and increases in spring reaching the maximum in autumn. This variation is characterized by the accumulation of neutral lipids, particularly free fatty acids and triacylglycerols, and probably reflects the annual cycle of feeding. The remarkably high ratio NL/PL in autumn was also observed in the digestive gland of males (Table 4) and the reproductive tissues (Jeckel et al., 1989a, b), and muscle of both sexes {Jeckel et al., 1991). This suggests that the shrimp would notably intensify feeding in autumn, after the peak of reproductive activity in summer (see below) and all of its tissues accumulate reserves, a part of which are probably used to undergo the winter. The fall of free fatty acids of the digestive gland in winter suggests that these components are consumed prior to triacylglycerols during periods of lower food
quality and/or availability (Table 5). The protein content in immature females, contrary to lipids, does not vary significantly during the year. The most striking results found in the females were the marked fall of all lipid components (excepting alkyldiacylglycerols) in the digestive gland of females $2 and $3, as well as the increase of protein concentration. A study on the biochemical composition of the stomachal content of P. muelleri collected from its natural habitat, showed that the shrimp does not interrupt feeding during the gonad maturation in summer (Jeckel et al., unpublished data). Thus, starvation as a cause of the lipid fall in females of summer can be discarded. On the other hand, the molting cycle of P. muelleri as in decapods in general, involves lipid consumption of the digestive gland from midpremolt to eedysis (see Jeckel et al., 1990). Maturing females, however, do not molt since growth and maturation are both energetically expensive and tend to be separated in time to avoid metabolic competition (Rice and Armitage, 1974; Adiyodi, 1985; Abell6, 1989). Besides, nearly 80% of females $3 used in this study had spermatophores attached to the thelycum (Jeckel et al., 1989a); these shrimps do not molt because it would imply the loss of spermatophores with the exuviae and consequently the failure of fertilization. Hence, the fall of lipids in the digestive gland of females $2-S 3 seems to be exclusively due to their utilization in the oogenesis process. The expression in absolute values can be suitable to describe the changes in biochemical composition of the digestive gland during oogenesis in summer. The calculations for a standard female (from Tables 1 and 3, and Jeckel et aL, 1989a) show that from stage Sm in spring to stage S2 in summer, the digestive gland loses 352 mg of lipid (with no selectivity in classes, Table 5) and accumulates 27.5 mg of protein whereas the ovary stores 27 mg of lipid and 129 mg of protein. Thus, the possible transference of lipids from the digestive gland to the ovary at early oogenesis would not explain the marked fall of lipids in the digestive gland of females $2. The fate of these lipids is not clear; perhaps they could be utilized at this stage for the synthesis in situ of ovary proteins. From early maturation ($2) until the complete ovary
Table 6. Seasonal variation in the concentration (mg/g of wet wt) of neutral lipid classes in the digestive gland of male shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 MRS stage
Winter S3
S1
Spring S3
Summer S3
Autumn S3
Low Rf components Free fatty acids Triacylglycerols Alkyldiacylglycerols
17.1 31.8 129.7 29.4
27.9 71.3 52.1 31.7
43.8 48.6 25.4 28.1
4.6 50.7 55.4 32.3
72.3 104.3 64.2 48.2
S~, immature reproductive system; S3, mature reproductive system. Low Rf components: partial glycerols, free sterols, pigments.
S e a s o n a l c o m p o s i t i o n o f shrimp---digestive g l a n d
257
Table 7. Relative per cent seasonal variation in the fatty acid composition of polar (PL) and neutral (NL) lipid fractions in the digestive gland of female shrimp, Pleoticus muelleri, San Jorge Gulf, 1984-1985 Winter SI PL NL Fatty acid 12:0 14:0 14:1 15:0 15:1 16:0 16:In - 7 16:2 + 17:0 16:3 18:0 18:ln - 9 18:2n - 6 18:3n - 6 18:3n - 3 20:1 (n - 9 + n - 11) 20:2n - 6 20:4n - 6 20:4n - 3 + 22:1 20:5n - 3 22:4n - 6 24:1 22:5n - 3 22: 6n - 3 Unidentified Saturated Monocnoie Polyenoic
Spring Sl PL NL
-2.9 1.7 2.1 2.9 19.8 13.2 -3.1 4.5 17.8 1.6 1.6 -4.0 2.1 3.6 -11.8 2.4 --4.8 --
1.0 3.2 1.5 1.7 1.7 17.5 15.2 1.6 2.6 4.1 20.1 0.9 1.2 -6.3 0.9 1.5 1.8 6.4 1.7 0.6 1.4 6.6 0.5
0.5 2.6 0.9 1.0 0.9 13.4 13.7 -1.9 2.4 17.4 1.4 1.4 0.7 7.2 1.4 2.1 1.8 14.0 1.0 0.8 1.8 11.7 --
0.8 3.3 1.0 1.3 0.9 16.8 13.3 1.5 2.0 4.4 20.7 1.4 1.5 -7.9 0.8 1.6 1.2 10.1 0.9 . 1.2 6.9 --
29.3 39.6 31.0
28.3 46.3 24.9
19.9 41.8 38.3
27.4 44.4 27.8
Summer PL
NL
PL
NL
Autumn $1 PL
-3.7 1.7 1.9 2.4 21.6 8.8 2.1 1.9 3.7 15.4 1.3 1.4 0.8 5.6 1.8 3.0 -9.2 2.0
-3.9 1.5 1.8 2.6 25.0 9.3 1.7 1.4 3.8 16.9 -0.5 -4.5 1.4 2.7 1.1 10.3 1.6
2.6 5.8 3.2
0.6 2.7 1.0 1.2 1.4 12.0 8.7 1.7 1.7 5.1 15.7 1.5 2.1 0.5 9.9 1.9 3.3 2.9 10.5 2.9 . 2.8 7.5 2.1
2.1 5.9 1.8
0.8 3.4 1.2 1.3 1.5 9.2 7.6 1.7 1.7 5.8 13.0 1.5 1.4 0.5 9.9 1.6 2.4 2.3 11.4 3.2 1.2 4.6 9.4 2.9
0.6 3.2 0.9 1.5 1.4 19.9 8.6 1.6 2.3 2.9 14.7 1.9 1.8 1.8 4.4 2.9 3.2 3.7 8.4 2.9 -1.3 8.6 1.4
0.7 3.4 0.9 1.2 0.8 16.5 10.7 1.2 1.7 3.2 18.3 1.8 1.4 1.3 6.3 1.3 1.7 3.3 8.8 1.0 -1.3 12.0 1.1
32.0 33.9 30.9
22.5 38.2 37.1
35.4 35.4 27.4
21.4 35.6 39.8
28.9 31.9 37.8
25.6 38.7 34.6
$2
.
.
$3
NL
S~, immature ovary; $2, incipient maturation; $3, complete maturity. - - , less than 0.5% or not detected.
development ($3) the digestive gland losses 37 mg of lipid and accumulates 33 mg of protein, whilst the ovary stores 120mg of lipid and 371 mg of protein. Thus, in this period the digestive gland contributes poorly with organic reserves to the developing ovary; consequently, the female shrimp would mainly
depend on its immediate food intake to satisfy the high vitellogenic requirements of triacylglycerols, phospholipids and protein (Jeckel et ai., 1989a). In the crab Carcinus maenas and the prawn Penaeus indicus the direct input of lipids from feeding were also preponderant (Heath and Barnes, 1970; Galois,
Table 8. Relative per cent seasonal variation in the fatty acid composition of polar (PL) and neutral (NL) lipid fractions in the digestive gland of male shrimp, Pleoticus muelleri, San Jorge Gulf, 1984--1985 Winter
Spring
S3
Summer
Si
S3
Autumn
S3
S3
PL
NL
PL
NL
PL
NL
PL
NL
PL
NL
Fatty acid 12:0 14:0 14:1 15:0 15:1 16:0 16:ln - 7 16:2 + 17:0 16:3 18:0 18: In - 9 18: 2n - 6 18:3n - 6 18:3n - 3 20:l(n - 9 + n - I1) 20: 2n - 6 20:4n - 6 20:4n - 3 + 22:1 20"5n - 3 22:4n - 6 24:1 22: 5n - 3 22:6n - 3 Unidentified
1.3 2.5 1.3 1.5 1.7 12.9 12.9 1.7 2.8 2.8 16.7 1.4 1.5 0.9 5.4 1.6 2.9 1.2 10.1 1.9 -1.9 11.4 1.6
1.3 3.1 1.4 1.7 2.0 14.4 12.9 1.8 3.0 4.3 18.7 1.2 1.6 1,0 7.2 1,3 2,3 1,6 7.0 1.5 -2.0 7.6 1.1
-3.6 1.1 1.3 1.1 13.6 12.1 1.3 2.0 2.9 16.4 1.6 1.4 0.7 7.2 1.6 2.0 2.4 13.5 0.7 -0.9 11.1 0.7
0.9 3.7 1.0 1.4 1.2 15.5 11.6 1.5 1.9 4.8 19.4 0.5 0.7 -7.7 0.7 1.9 1.9 11.7 1.0 0.6 1.3 8.7 --
0.8 3.8 !.0 1.2 1.0 13.6 12.2 1.3 2.3 3.2 16.8 1.3 1.5 0.7 7.0 1.4 2.1 1.5 13.8 1.5 0.9 2.0 9.1 --
-4.1 1.0 1.3 1.0 15.9 11.6 1.5 2.0 5.2 18.6 1.5 1.3 -8.5 1.3 1.9 -11.5 1.0 0.8 1.5 7.4 1.0
0.5 2.7 0.9 1.1 0.9 10.4 7.8 1.3 1.7 3.4 14.4 1.3 1.1 0.8 7.5 1.7 3.5 1.6 15.3 2.5 0.7 3.2 14.7 1.0
1.1 3.5 1.1 1.4 l.l 16.1 8.5 1.7 1.5 6.3 19.8 1.4 1.3 0.6 8.5 1.6 2.0 1.8 7.7 1.4 -1.8 5.3 4.5
-1.3 1.7 1.7 1.3 22.2 8.8 1.3 1.9 3.0 17.6 0.6 -0.9 5.0 1.0 1.9 3.0 9.9 ---11.8 1.4
-3.9 0.9 1.3 0.8 16.9 10.3 1.2 1.8 3.9 19.4 1.4 1.3 1.2 6.4 1.2 1.6 3.0 9.4 0.5 0.5 0.6 12.4 --
Saturated Monoenoic I: Polyenoic
21.9 38.6 37.9
25.7 43.0 30.2
22.1 39.4 37.4
27.1 42.5 30.2
23.2 39.6 37.2
27.3 41.5 30.2
18.8 33.0 47.3
29.3 39.9 26.4
28.9 35.9 30.2
26.6 39.8 33.5
S~, immature reproductive system; $3, mature reproductive system. - - , less than 0.5% or not detected.
258
WALTERHORACIOJECKELet al.
1984). In other decapods, however, the contribution in organic reserves of the digestive gland to the developing ovary was more important (Allen, 1972; Clarke, 1977; Kulkarni and Nagabhushanam, 1979; Teshima and Kanazawa, 1983). Protein storage in the digestive gland during oogenesis could be due to a decrease in the proteolytic activity of this organ in order to maintain an alternative source of energy. The scarce lipid supply from the digestive gland to the ovary, from $2 to $3, consisted of free fatty acids, triacylglycerols and phospholipids, whereas the increase of the low Rf components in the digestive gland was probably due to an accumulation of hydrolysis products (Table 5). P. muelleri appears to be lesser selective than the prawn Penaeus japonicus as the latter only transferred triacylglycerols from the digestive gland to the ovary during vitellogenesis (Teshima and Kanazawa, 1983). The fatty acid composition of the digestive gland is typical of marine decapods, with high levels of acids 16: 0, 16: In - 7, 18 : In - 9 and a marked predominance of the n - 3 polyenoic acids, mainly 20: 5 and 22:6 (Addison et al., 1972; Gopakumar and Nair, 1975; Guary et al., 1975; Middleditch et al., 1980; Chapelle, 1986). Fatty acid 20:1 reaches higher levels in the digestive gland than in the remainder of shrimp tissues (Jeckel et al., 1989a, b, 1991) as appears to be a common feature in several decapods (Allen, 1971; Teshima and Kanazawa, 1983). The fatty acid composition varied in both polar and neutral lipids of the digestive gland throughout the year. However, total fatty acid composition should reflect that of neutral lipids as this fraction is quantitatively more important (Tables 3 and 7). The most notable changes in the fatty acid composition of the digestive gland occur in summer during the ovary development. Saturated and monoenoic fatty acids of neutral lipids decrease with the progress of oogenesis, due to the apparent selective utilization of acids 16:0, 16:1 and 18:1; consequently, the level of polyenoic acids increases. Jeckel et al. (1989a) found a significant increase in the level of palmitic acid (16:0) in the ovary of P. muelleri during vitellogenesis, but not so of acids 16:1 and 18:1. Although palmitic acid is efficiently synthesized de novo by crustaceans (Castell, 1981), it cannot be ignored that at least in part this acid is transferred from the digestive gland to the developing ovary, whereas the acids 16:1 and 18:1 could be consumed as a source of energy. Teshima and Kanazawa (1983) found no significant variations in the fatty acid composition of the digestive gland and ovary during vitellogenesis of P. japonicus, thus suggesting that the fatty acid metabolism during this process varies considerably from one species to another. It is interesting to note that the ratio 22: 6/20: 5 in neutral lipids of the digestive gland and ovary varies with a similar pattern throughout the year. The origin of these fatty acids in the shrimp and the possible causes of their variations in relation to food quality have been discussed in previous studies (Jeckel et aL, 1989a, b). The variation in the fatty acid composition of polar lipids showed no clear seasonal trends. The major
part of these lipids would not be biomembrane components of the digestive gland but possibly reserves, judged by the marked variations in their contents (Table 3) and fatty acid levels. These variations could be due to diverse factors acting simultaneously, such as the dietary input, turnover, and the lipid transport mechanisms of the shrimp. Males
The relationship between the digestive gland weight and the total body weight is more variable in males than in females, since in males $3 of spring and autumn no correlation was found (Table 2). Although the relative size of the digestive gland of males as well as in females decreases in summer, the concomitant changes in the biochemical composition of this organ differ remarkably between sexes as is discussed below. The amount of lipid in males $3 increases from winter toward the autumn (except in summer, see below) which reflects the seasonal dietary changes discussed in females (Tables 3 and 4). However, the ratio NL/PL in males, as opposed to females, decreases from winter to summer with a striking increase of polar lipids. This suggests that the lipid metabolism of the digestive gland differs considerably between sexes during the year. Phospholipids are generally recognized because of their roles in transport processes and in the structural and functional maintenance of biomembranes (Chapelle, 1986). However, the remarkable variation in phospholipid content in the digestive gland of males suggests again a role as reserves in P. muelleri. Phosphoglycerides as depot lipids have also been proposed in adult krill Euphausia superba (Saether et al., 1985) and fish larvae (Tocher et al., 1985). In winter, the neutral lipids of males $3 (similarly to females) are rich in triacylglycerols, with the suggestion that the digestive ~ gland would utilize mainly free fatty acids during periods of low food availability (Table 6). In spring, the lipid composition of immature males (SI) differs markedly from that of mature males ($3). The fall of lipids in the digestive gland from Sl to S3 appears to be exclusively due to their utilization in the spermatogenesis process. The calculations for a standard male (from Tables 2 and 4) show that from stage S1 to S3 the digestive gland losses 65 mg of neutral lipids and 24 mg of polar lipids, whilst the protein content does not vary significantly. The lipid content of the reproductive system (testes plus spermatophores) does not vary appreciably with maturation whereas total nitrogen content increases from 29.4 to 35.3 mg (Jeckel et al., 1989b). This would suggest that nitrogenous compounds of the reproductive system are synthesized in situ during spermatogenesis, at least in part at the expense of the digestive gland. Free fatty acids and triacylglycerols (Table 6) would be utilized as a source of energy whereas phospholipids could be involved in transport processes. The results expressed in terms of concentration may lead to erroneous conclusions when values of an organ changing its size are compared. This may occur with the shrimp in summer, when the percentage values of biochemical components are high but the digestive gland size is around one-fifth smaller than
Seasonal composition of shrimp--digestive gland in the other seasons. In absolute amounts a standard male in summer (DGI = 3.99) would contain 108 mg of lipid and 44 mg of protein less than a hypothetic male (in summer) with a DGI = 5.32 (average index of males $3 of winter, spring and autumn). These morphometric and biochemical changes in the digestive gland of male shrimp in summer could indicate symptoms of starvation. It has been observed that starved marine invertebrates have the capacity to resorb body tissues and thus utilize biochemical components without affecting their levels (see Lawrence, 1976). It is worth mentioning a preliminary survey during a breeding season that showed stomachal content in male shrimp had 50% lesser lipid than that of females, thus suggesting energetically lower food in the formers in this period (Jeckel et al., unpublished data). The results of the present study would agree with those observations and lead us to postulate that males may undergo a metabolic stress in summer with the consequent degradation of tissues in the digestive gland. It is interesting to note that in spite of the morphometric changes, the absolute amount of polar lipid remains fairly constant between spring (DGI = 5.08) and summer (DGI = 3.99). This would suggest that the shrimp tends to conserve phospholipids in the digestive gland. The glycerophospholipid content could be maintained at the expense of partial glycerols (Table 6). Interestingly, Heath and Barnes (1970) noted the rise in phospholipid possibly at the expense of neutral fat in starved Carcinus maenas, but they could not explain the causes of such increase. In the case of the shrimp, phospholipids would appear to be reserved for some specific process probably occurring after the breeding period. Neutral lipid consumption may be accompanied by cell destruction with the consequent metabolization of protein, although utilization of non-protein nitrogenous compounds cannot be totally discarded. Possibly, males change their feeding habits, eat little, and increase locomotory activity during mating in summer; consequently, their energetic requirements would exceed the dietary input, as occurs in other species (Armitage et al., 1972; Tsai et al., 1984). It is concluded that, although the relative size of the digestive gland decreases in mature shrimps ($3) of summer, the causes of this morphometric variation as well as the changes in biochemical composition, differ remarkably between sexes. In autumn, the ratio NL/PL reaches the maximum value and is very similar in both sexes (Tables 3 and 4). Luxuriant feeding in this season appears to be the preponderant factor determining the lipid composition of the digestive gland, characterized by the accumulation of neutral lipids (Tables 5 and 6). The fatty acid spectrum in the digestive gland of both sexes, as well as the similar pattern in the ratio 22: 6/20:5 of neutral lipids, suggest an important influence of diet throughout the year. However, males had scarce variations in the level of major fatty acids, except in polar lipids in summer (Table 8). In this season, the increase in level of polyenoic fatty acids is apparently due to a selective utilization of the acids 16: 0, 16:1 and 18 : 1. Thus, the decrease in size of the digestive gland would not involve the loss of polar lipids (Tables 2 and 4) but a qualitative change in this fraction. With the fall of polar lipids in autumn the
259
digestive gland losses substantial amounts of polyenoic fatty acids, which are apparently replaced by palmitic acid (16:0). Such variation of fatty acids is not observed in females, thus suggesting that the causes would not be of a dietary origin and, at least in part, palmitic acid could be synthesized de novo by males. It has been observed for crustaceans and fish that chain elongation and desaturation of fatty acids tend to increase as a response to low environmental temperatures (Castell, 1981). The water temperature in the natural habitat of the shrimp (at a depth of 36-86 m) ranges from 6.7°C in winter (August) to 13.9°C in summer (January) (Jeckel et aL, 1989a). Consequently, an increase of polyenoic fatty acids could be expected to occur in winter, but this is not found to be the case. Probably, the fatty acids of dietary origin and their accumulation or selective consumption are preponderant in determining the lipid composition in the digestive gland of the shrimp. Acknowledgements--The authors are indebted to Lic. A. Ptrez for assistance in statistical calculations, and to Lic. S. De Marco who revised the translation of the manuscript. REFERENCES
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