Comp. Biochem. Physiol. Vol. 96B, No. 3, pp. 505-511, 1990 0305-0491/90 $3.00 + 0.00 Printed in Great Britain Pergamon Press plc

THE BIOCHEMICAL COMPOSITION OF EGGS FROM MACROBRACHIUM ROSENBERGH IN RELATION TO

EMBRYONIC DEVELOPMENT

ANDREW CLARKE, JANET H. BROWN* and LESLEY J. HOLMES

British Antarctic Survey, NERC, High Cross, Madingiey Road, Cambridge CB3 0ET, England, UK; and *Institute of Aquaculture, University of Stifling, Stirling FK9 4LA, Scotland, UK

(Received 24 November 1989)

Abstract--1. The proximate (water, protein, lipid, mineral ash) and elemental (carbon, nitrogen, phosphorus) composition of eggs of the freshwater prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea, Decapoda) were determined in relation to embryonic development. Eggs were sampled at four intervals during development, ranging from day I (newly spawned) to day 16 (about to hatch).

2. Egg dry mass, lipid and carbon content all decreased during development as reserves were utilized; at the same time both water and mineral ash content increased. Although the mean content of both nitrogen and protein declined during development these were not statistically significant due to a high variance. Egg phosphorus content remained constant throughout development.

3. In the lipid fraction triacylgycerol fatty acids provided the major fuel, although phospholipid was also utilized. The most important substrates were 16:0 and 18:1 fatty acids.

4. There were strong suggestions of an initial increase in egg lipid content during the first 5 days of development, though this was not statistically significant and the source of the extra material is unknown.

INTRODUCTION

The eggs of aquatic invertebrates range widely in size. Even within a single taxonomic group such as deca- pods or amphipods, egg size can vary enormously between species, and also within species. For example echinoderm eggs vary in diameter from 50 to 1500/~m (Turner and Lawrence, 1979) and in caridean deca- pods egg size ranges from 50/~g dry mass (0.5 mm diameter) in typical warm-water species to 3 mg dry mass (3.4 mm longest dimension) in Lebbeus antarcti- cus (Clarke, unpublished data). Within species differ- ences include latitudinal variations within different parts of a species' range, or seasonal differences within an extended breeding season. In general, of course, species with smaller eggs have higher fecundi- ties than those with larger eggs, and the selective advantages of different egg sizes have been discussed extensively in the literature of marine invertebrate reproduction (e.g. Vance, 1973a,b; Christiansen and Fenchel, 1979; Perron and Carrier, 1981).

Associated with these variations in egg size are differences in the time taken for the embryo to develop and hatch. This can vary from a few days in some tropical species to at least 18 months in some polar isopods (Luxmoore, 1982; Wagele, 1987). In general larger eggs take longer to hatch than smaller eggs, due in part to the larvae developing to a more advanced stage before being released. It is therefore of interest to know whether there are any differences in chemical composition or the pattern of yolk utiliza- tion between large and small eggs.

We have undertaken a study of changes in the biochemical composition during development of the eggs of the warm water decapod prawn Macro-

brachium rosenbergii (de Mann, 1879), to compare with data available for temperature and polar species. Of particular interest is that in at least one polar species, the caridean shrimp Chorismus antarcticus, the initial period of embryonic development appears to involve an increase in egg lipid content before the more usual pattern of lipid utilization (Clarke, 1977). This is a most unusual result, although there are some indications of an initial increase in egg lipid content during incubation in some other polar crustaceans (see discussion in Clarke et al., 1985). In this study of Macrobrachium rosenbergii we therefore attempted to answer three questions:

1. What are the major fuels for embryonic development?

2. What are the changes in lipid class and fatty acid composition during embryonic development?

3. Is there an increase in egg lipid content in the early stages of incubation?

MATERIALS AND METHODS

The original brood stock of Macrobrachium rosenbergii was obtained from Thailand and held in culture at Stirling. Females in this study were of the F1 generation. Prawns were reared communally in a recirculated water system at 28 _ I°C. They were fed daily with a pelleted diet (40% protein based on squid, shrimp and fish meal, and produced at the Institute of Aquaculture), supplemented with fresh food (mussels, prawns, fiver and fish flesh) initially twice weekly but then daily once maturity was attained. Mature females were kept individually and checked daily; if they had newly moulted a male was introduced to the tank for 2 hr. Eggs were spawned later the same day, day 0 of the incubation period.

505

506 ANDREW CLARKE et al.

At intervals during incubation complete egg batches were collected for analysis as follows:

Day 1 Sample I Five individuals 5 Sample 2 Five individuals

10 Sample 3 Five individuals 15 Sample 4 Four individuals 16 Two individuals 17 Two individuals.

At 28°C eggs generally hatch about day 17. In most analyses relatively little difference could be detected between eggs sampled on days 15, 16 and 17. The data for these eggs were therefore combined into a sample of eight individuals assigned to day 16 (Sample 4).

Size and mass of an individual egg

The complete egg sample was weighed immediately. A subsample was then removed, weighed, and preserved in 4% formalin. The remainder of the sample was frozen to -20°C for later analysis. In Cambridge the formalin-preserved sample was reweighed and counted under a stereomicro- scope; this provided an estimate of the wet mass of an individual egg. The long and short axes of a sample of 20 eggs from each sample were measured with a video camera coupled to a computer image analyser, and egg volume calculated from the formula for an ellipsoid.

Chemical composition

All chemical analyses were performed on subsamples of the frozen eggs. Dry mass and water content were deter- mined after drying a subsample for 48 hr at 60'~C. This dried material was then used for the determination of ash, carbon, nitrogen and phosphorus. Ash content was estimated after ignition for 12 hr in a furnace at 500°C. Carbon and nitrogen were measured with a Carlo Erba 1106 elemental analyser; samples were analyzed in duplicate, with acet- anilide as calibration standard. Phosphorus was measured as orthophosphate according to Bartlett (1959), following digestion with 72% perchloric acid for 30 min at 180°C.

Protein and lipid analyses were also performed on weighed subsamples of the frozen eggs, but without drying since this is deleterious to both assays. Protein was assayed according to Lowry et al. (1951) as modified by Hartree (1972), using bovine serum albumin as a standard. Lipid was extracted by homogenizing in methanol/chloroform (Bligh and Dyer, 1957); the lipid content was estimated gravimet- rically after transferring the extract to a preweighed vial and evaporating the solvents with a slow stream of oxygen-free dry nitrogen. Lipid class composition was estimated by scanning densitometry following separation of the lipid classes by high performance thin-layer chromatography (TLC). Triacylgycerol and phospholipid were isolated for fatty acid analysis by preparative TLC, and fatty acid methyl esters (FAMEs) prepared by reaction with NaOH in methanol. The FAMEs were purified by column chromatog- raphy on silica gel and taken up in hexane. They were then analyzed by gas chromatography on a 25 m fused silica capillary column coated with Carbowax 20M in a Hewlett Packard 5792 GC, using helium as carrier gas. Samples were injected in splitless mode and the oven temperature pro- grammed from 175 to 210°C during analysis. FAME peaks were integrated electronically using a HP3390A integrator. For details of analyses, identification and data handling see Clarke et al. (1985).

RESULTS

Egg volume and mass

The volume, wet mass and dry mass of an individ- ual egg at different stages of deve lopment are given in Table 1. Between day ! and day 16 egg volume increased by 50% and egg wet mass by 30%. This

increase in volume was due solely to uptake of water, for over the same period dry mat te r decreased by 20% as reserves were utilized. Egg water conten t thus increased f rom 61/~g (55% of the wet mass) to 106 p g (72% wet mass) dur ing development .

The egg volume and mass data reported in Table 1 suggest a mean density of 2.0 for an egg at day 1. This is quite a high value. The size of a day 1 egg preserved in formal in (about 480 pm) is also smaller than that reported for a mature oocyte (about 530pro) by O ' D o n o v a n et al. (1984). There is no indicat ion of any significant change in egg mass dur ing preserva- t ion (see below), and so it seems likely tha t the eggs shr ink slightly once they have been extruded.

Precision o f the composition data

Egg composi t ion data are often reported as a funct ion of the dry mass or wet mass. However if the size and mass of the egg change dur ing development as well as the chemical composi t ion (as happens here with Macrobrachium rosenbergii and is usual for mar ine inver tebra te eggs), unless composi t ion data are expressed as a m o u n t per individual egg then impor t an t changes may be missed. Wi th large eggs (such as those typical of many polar crustaceans) this is s t ra ightforward, for eggs can be counted before being preserved or analyzed. Wi th eggs as small as those f rom Macrobrachium it is no t possible to count eggs either before or after freezing. The n u m b e r of eggs in a frozen sample must therefore be es t imated from the mass of tha t sample and the mass of an individual egg. This involves the assumpt ion tha t the mass of an egg preserved in formal in (for count ing and weighing) and that of an egg frozen for analysis are the same. In this study the rat io of the mass of the subsample before and after preservat ion in formal in was 1.12 (SD + 0.14). There was no significant varia- t ion in this rat io with development t ime (one-way A N O V A , F = 1.65, P > 0.05), and the mean ratio was not significantly different f rom unity (t = 0.85, P > 0.05). The assumpt ion was thus a reasonable one.

A fur ther p rob lem is tha t when the mass of an egg is small compared with the size of the frozen sample, the precision of the est imate of the n u m b e r of eggs in tha t sample is low. Nevertheless with a species such as Macrobrachium this is the only feasible technique. It is inevitable, however, tha t the wide confidence intervals a t tached to some of the estimates of bio- chemical componen t s discussed below are due in par t to errors in est imating egg number .

Table 1. Volume (nl), wet mass and dry mass (both in #g/egg) of individual eggs of Macrobrachium

rosenbergii

Volume Wet mass Dry mass Day n (nl) (~g) (#g)

I 5 56(4) 112(25) 50.7(12.1) 5 5 65(10) 118(14) 52.0(5.8)

10 5 59 (2) t34 (14) 49.8 (6.0) 16 8 84(17) 147(21) 40.9(6.1) F 8.14 4.11 2.93 P < 0.001 <0.018 NS (0.056)

Data are means, with SD in parentheses. n = number of individual prawns analyzed. F = variance ratio for one-way ANOVA against

day. P = two-tailed probability of this result. NS = not significant (P > 0.05).

Macrobrachium egg composition 507

Elemental composition The content of carbon, nitrogen, phosphorus and

total mineral ash in individual eggs in relation to development time are shown in Fig. 1. Phosphorus content was generally low (0.5 #g/egg or less) and showed little variation. Nitrogen content was gener- ally 4--5 #g/egg and tended to decrease slightly during the later developmental stages. Mineral ash increased throughout development, presumably as calcium was accumulated for the exoskeleton. The most dramatic change was, however, shown by carbon which de- creased from 29.5 #g/egg at day 1 to 21.8 #g/egg at day 16, a decrease of just over 25%. Carbon was therefore providing the main fuel for development, as would be expected. Of these changes, only those involving carbon and mineral ash were statistically significant (Table 2).

Lipid and protein content The lipid and protein content of individual eggs in

relation to development time are shown in Table 3. There was a significant variation in lipid content, with a particularly marked drop between day 10 and day 16. There was also an indication of a slight increase in lipid content in the first 5 days of development, but this was small in relation to the variance. Interest- ingly a similar small increase was also detectable in protein content, although overall the variation in protein was too little to be statistically significant (Table 3). The energy content of the eggs inevitably showed a similar pattern, since this was calculated from lipid and protein content rather than estimated by direct calorimetry (the carbohydrate content of the eggs was too small to influence the calculated energy content significantly--see below).

Was this initial increase in lipid and protein real, or an analytical artefact? The mean contents of both carbon and nitrogen (which were analyzed com- pletely independently from lipid and protein) showed an increase between days 1 and 5, although the

30

A 25 $

20 |

5

$ • Ash

0 ~'-"'~ o o o P

O ' 5 110 1'5 I 117 Development Time (daYlz)

Fig. 1. Carbon (C), nitrogen (N), phosphorus (P) (a]] O ) and mineral ash (O) content o f an indiv idual egg o f Macrobrachium rosenbergii during embryonic development. All data expressed as #g/egg, and plotted as mean with 95% confidence interval (2 x SE of the mean); only one error bar being shown for clarity. Where none is shown, the error bar is smaller than plotted symbol, n = 5 (days 1 to 10) or n = 8 (day 16), where n is the number of separate egg batches analyzed. Note displacement of vertical axis, and change of

scale for carbon data.

Table 2. Variation in carbon (C), nitrogen (N), phosphorus (P) and mineral ash content (all in gg) of an individual egg of

Marcobrachium rosenbergii in relation to development

Day n C N P Ash

I 5 29.5 (7.0) 4.83 (1.22) 0.50 (0.1 I) 0.90 (0.28) 5 5 29.9 (3.3) 5.05 (0.73) 0.47 (0.08) 1.74 (0.60)

10 5 27.9 (3.3) 4.74 (0.52) 0.50 (0.10) 2.29 (0.24) 16 8 21.8 (3.4) 4.16 (0.60) 0.43 (0.07) 2.31 (0.32) F 4.96 1.59 0.95 17.01 P 0.008 NS NS < 0.0001

Presentation and abbreviations as for Table 1.

confidence intervals were high for carbon and the overall variation in nitrogen was not statistically significant. Against this must be set the high variance in most measures (and particularly protein) at day 5. The evidence for an initial increase in lipid and protein content is thus suggestive but far from conclusive.

Taken together, the protein, lipid and mineral ash fractions explained on average 101.4% (SD 16.7) of the total dry matter. The 95% confidence intervals on the mean value of total explained matter were 94.2 and 108.6% of the measured dry mass. This indicates that components such as carbohydrate, nucleic acids, and intermediary metabolites probably total less than 5% on average. The steady, small (but statistically non-significant) decrease in nitrogen content may represent excretion of ammonia and/or urea during embryonic metabolism.

Lipid class and fatty acid composition As is typical of crustacean eggs the lipid consisted

primarily of phospholipid (PL: 28% of the total lipid at day 1) and triacylglycerol (TAG: 71% at day 1). There were also small amounts of free sterol ( < 1%), together with traces of free fatty acid and partial glycerides. A compound of high TLC mobility (which may have been sterol ester) occasionally formed up to 2% of the total lipid. Thin-layer chromatography indicated that most ( > 50%) of the PL was phos- phatidyl choline, but variations in phospholipid com- position with development were not examined.

The relative proportions of the two major lipid classes, PL and TAG, changed slightly during embry- onic development. However a much clearer picture is seen if the data are converted to show the absolute amount of PL and TAG per individual egg (Fig. 2). Phospholipid showed a steady decline throughout development, though utilization was fastest between days 10 and 16 Triacylglycerol decreased during the later stages of development (days 5 to 16); there were

Table 3. Variation in the proximate composition (lipid, protein, #g), and energy content (J) of an individual egg from Macrobrachium rosenbergii in relation to

development

Day n Protein Lipid Energy

1 5 31.1 (5.3) 14.4(3.6) 1.31 (0.24) 5 5 36.7 (I 3.0) 14.7 (2.7) 1.46 (0.34)

10 5 34.4 (7.1) 13.7(2.1) 1.14 (0.12) 16 8 31.9 (8.5) 9.4 (1.7) 1.14 (0.24) F 0.43 6.82 1.97 P NS 0.002 NS

Energy content was calculated assuming standard enthalpies of combustion (Gnaiger and Bitterlich, 1984).

Presentation and abbreviations as in Table I.

508 ANIge~w CLARKE et al.

12

4

~ T A G

~ - - 5 - - 10 - - 1'5 ~-1J7 Development Time (days)

Fig. 2. Triacylglycerol and PL content of an individual egg of Macrobrachium rosenbergii during embryonic develop- ment. Data calculated from lipid class composition data and total lipid content. All data expressed as #gjegg, and plotted as mean with 95% confidence intervals (2 x SE). n = 5 (days l to 10) or n = 8 (day 16), where n is the number of separate egg batches analyzed. Note displacement of vertical axis for

clarity.

indications of a slight increase in T A G between days 1 and 5 but this was not statistically significant for the confidence intervals were wide. The overall variations in both PL and T A G were statistically significant (one-way A N O V A , PL: F = 4.24; TAG: F = 4.69; both P < 0.05). The differing patterns in Fig. 2 thus suggest that if there was indeed an increase in lipid between days 1 and 5, this was due to an increase in T A G and not PL.

The major features of the fatty acid composit ion of PL and T A G isolated from eggs at day 1 are shown in Table 4. Al though the proport ions differed, the same fatty acids dominated the composit ion

Table 4. Fatty acid composition of PL and TAG isolated from newly spawned (day 1) eggs of Macrobrachium rosenbergii

Fatty acid PL TAG 14:0 1.8 3.7 16:0 19.8 26.4 16:1 (2 isomers) 6.1 8.8 18:0 12.4 6.0 18:1 (4 isomers) 25.3 25.1 18:2 (n-6) 3.6 4.5 18:3 (n-3) 1.9 2.6 18:4 (n-3) 1.2 1.5 20:1 (2 isomers) 3.2 2.1 20:4 (n-6) 2.9 2.1 20:5 (n-3) 8.5 5.7 22:1 (2 isomers) hi 0.8 22:5 (n-3) 1.3 0.9 22:6 (n-3) 5.0 4.5

minor components* 5.1 6.0 r unidentified FAMEs (n) 1.2 (19) 1.2 (22) Z" saturated FAMEs 35.5 36.6 L" monoenoic FAMEs 36.4 37.6 ,r polyenoic FAMEs 22.3 19.0 Unsaturation (db/mol) 1.51 1.36 Mean C chain length 17.96 17.57 *Minor components were 15:0, 17:0, 20:0, 15:1, 17:1, 24:1, 16:2

(n-6), 20:2 (n-6), 16:3 (2 isomers), 18:3 (n-6), 20:3 (2 isomers), 20:4 (n-3), 20.5 (n-6) and 22:5 (n-6).

Mean CV for major FAMEs was + 19.9%, and for minor FAMEs was +_ 35.9%.

Data presented as a percentage of the total uncorrected detector response for all FAMEs comprising > 1% total in either PL or TAG.

Each value is the mean of three separate batches of eggs analyzed individually.

db/mol: mean number of double bonds per FAME molecule.

of both PL and TAG, namely 16:0, 18:0 and 18:1. All generally formed > 15% of the total fatty acids (although 18:0 was only 6% in TAG). Important other components were 16: 1, 20:5 and 22:6, and there were smaller amounts of 14:0, 18:2, 18 : 3, 18 : 4, 20:1, 20: 4, 22 : 1 and 22: 5. As is usual in fatty acid mixtures isolated from aquatic organisms, analysis by capillary column revealed a large number of minor isomers and other fatty acids at the trace level and below (see details in Table 4). These, however, are not known to be of biological impor- tance in terms of embryonic development and so have not been discussed further here. Overall the fatty acids isolated from PL were of longer chain-length and were more unsaturated than those from T A G (Table 4).

In both PL and T A G there were roughly equal proportions of saturated and monoenoic fatty acids. However, the proport ion of polyenoic fatty acids (3 or more double bonds) was lower in T A G (19.0%) than in PL (22.3%: Table 4). In T A G all three fractions were utilized throughout embryonic devel- opment (Fig. 3). In PL, although all three types of fatty acid were utilized in the first 5 days of develop- ment, after this time utilization of monoenoic and polyenoic fatty acids slowed relative to that of satu- rated fatty acids.

A clearer indication of the relative importance of these three fatty acid fractions can be obtained from a simple budget calculation (Table 5). Since both PL and T A G are utilized as fuel during develop- ment (Fig. 2) data for fatty acids from both lipid classes have been combined. In absolute terms the most important fuels were clearly 16:0 and 18:1, which is not surprising since these were the two major fatty acids present. In terms of propor- tional utilization, however, (i.e. how much of the fatty acid originally present in the egg was used as fuel) saturated fatty acids were most conserved (36% used), followed by monoenoics (42% used)

oL 6

= -- Set t ~'~ Poly

= 5 1~0 15 17 Development Time (days)

Mono Sat

Mono

Fig. 3. Utilization of fatty acid classes during embryonic development in Macrobrachium rosenbergii. Data are for saturated (Sat), monoenoic (Mono) and polyenoic (Poly) fatty acids, plotted separately for TAG (C)) and PL (0). Data were calculated from fatty acid composition data and total amount of TAG and PL at each stage. All data expressed as #g/egg, and plotted as mean with 95% confi- dence intervals (2 × SE calculated from the variance of the fatty acid analyses only). Only one error bar is plotted for clarity, and where none shown the error bar is smaller than plotted symbol, n = 3 (days 1 to I0) or n = 6 (day 16), where n is the number of separate egg batches analyzed. Note

displacement of vertical axis for clarity.

Macrobrachium egg composition 509

Table 5. Utilization of different fatty acids during embryonic development of Macrobrachium rosenbergii

Mass FA (/~g/egg) (PL + TAG) Day n 16:0 18:0 16:1 18:1 20:4 20:5 22:6

I 3 3.50 1.03 1.21 3.70 0.31 0.91 0.67 16 6 2.26 0.65 0.67 2.28 0.19 0.42 0.25 Utilized 1.24 0.38 0.53 1.42 0.12 0.49 0.42

(~g) Utilized 35.5 36.6 44.1 38.4 39.1 54.3 62.5

(%) Data for seven major fatty acids (FAs) calculated separately for PL

(2 mol FA/mol) and TAG (3 tool FA/mol) and then combined to provide a total mass and presented as/~g FA/egg.

Amount utilized calculated from that present at days 1 and 16, and presented both as mass utilized (/~g) and as a percentage of that present at day 1.

n, number of separate egg batches analyzed individually to provide data for that day.

and then polyenoics (52% used). As a result the embryo fatty acids were less unsaturated than those supplied in the yolk. There is thus no evidence whatsoever of conservation of polyenoic fatty acids during development (for use in membranes, for example). The relatively low utilization of 20:4 (n-6) compared with 20:5 (n-3) and 22:6 (n-3) does suggest the possibility of a degree of conservation of (n-6) fatty acid (as was also indicated in gammarid amphipod eggs: Clarke et al., 1985).

DISCUSSION

Egg composition

The biochemical composition of eggs of Macro- brachium rosenbergii was typical of crustaceans. At spawning the eggs has a low water content (55% wet mass) and a low ash content (0.9% dry mass); the remainder of the egg comprised a protein- rich yolk. Taken together the protein and lipid con- tents explained virtually all of the organic content of the egg. The balance will consist of a small amount of carbohydrate, nucleic acids and intermediary metabolites. If the N content of yolk protein is taken to be 16% (the usually accepted value for a general- ized protein), then the observed protein content requires between 105 and 110% of the observed N content, depending on the stage of development. Some N is present in PLs, macromolecules and many intermediary metabolites of small molecular weight. Clearly although the pattern of N content with development is dictated largely by variation in protein content (the two measures vary together: Tables 1, 2), yolk protein in Macrobrachium contains only about 14% N.

The lipid fraction of newly spawned eggs was predominantly TAG (71%) and PL (28%). Typically most of the fatty acids were either saturated or monoenoic, suggesting a significant contribution of synthesis de novo in addition to dietary input during maturation of the eggs in the ovary (Clarke, 1979; Clarke et aL, 1985). The polyenoic fatty acid fraction was smaller than in the yolk of larger crustacean eggs. Long chain polyenoic fatty acids are usually regarded as essential dietary components in crus- taceans, which are believed not to be able to synthe- size these de novo. There may thus be a greater requirement for an initial supply of polyenoic fatty

acids in the yolk of larger eggs with longer develop- ment times (Clarke et al., 1985).

Changes in composition during development

The eggs of Macrobrachium rosenbergii are small and contain only 45/zg yolk when spawned. Embry- onic development takes 17 days, after which time a zoea hatches. Immediately before hatching (day 16) eggs still contained about 41/~g lipid plus protein, equivalent to nearly 90% of the energy originally present in the egg; this suggests that plenty of reserves were left at hatching. The newly hatched zoeae do not feed and the remaining yolk is consumed within 24 hr (Goodwin and Hanson, 1975).

Although both lipid and protein were utilized during the later stages of development, lipid provided the greater amount of energy (0.21 J as against 0.12J from protein between days 5 and 16). The ratio between protein and lipid in the egg thus changed from 2.15 at day 1 to 3.38 at day 16. In Crangon crangon both protein and lipid were utilized but lipid was the major fuel both in terms of mass consumed and energy provided (Pandian, 1967).

Both TAG and PL were utilized during development, but the bulk of the fuel was provided by TAG (3.7 #g TAG between day 1 and day 16 as against 1.4/zg PL: Fig. 2). Allowing for the difference in fatty acid content (2 mol/mol in PL, 3 mol/mol in TAG) indicates that of the fatty acid fuel utilized during development, roughly 75% was provided by TAG and 25% by PL. Although some PL must be retained for cellular membranes in the developing embryo, the use of yolk PL for fuel has been demon- strated previously in crustaceans (Clarke et al., 1985) and fish (Tother et al., 1985).

The major fatty acids utilized during development were 16: 0 and 18:1 (Table 5), and although there was a high percentage utilization of polyenoic fatty acids (primarily in TAG) these provided relatively little energy in absolute terms (Fig. 3). The pattern of percentage utilization with polyenoics > monoenoics > saturated is similar to that found in two species of gammarid amphipods (Clarke et al., 1985). The lower rate of utilization of polyenoic fatty acids in PL may reflect some conservation for membranes in the devel- oping embryo (although the overall pattern of utiliz- ation gives little support for this), and there was also equivocal evidence of selective conservation of (n-6) fatty acids relative to (n-3).

Comparison with other species

There are surprisingly few data from other species of crustacean with which to compare this study. In Crangon crangon the eggs are smaller than in Macro- brachium (16.6/~g dry mass at spawning: Pandian, 1967) but development time is much longer (7-10 weeks in the Bristol Channel: Lloyd and Yonge, 1947). The eggs contain 9.8 #g protein and 5.4/~g lipid at spawning, but lipid provides the major fuel. In the polar carideans Chorismus antarcticus, Notocrangon antarcticus and Nematocarcinus lon- girostris the eggs are much larger and protein again is the major reserve (Clarke, in prep.). These eggs take at least 10months to develop and the pattern of utilization is largely unknown.

510 ANDREW CLARKE et al.

An initial increase in lipid content?

The data for dry mass (Table 1), carbon, nitrogen (Table 2, Fig. 1), protein and lipid (Table 3) all suggest that there was an increase in egg nutrient content after spawning. This occurred between day 1 and day 5, after which the more expected pattern of steady utilization of reserves was seen. The data on lipid class composition (Fig. 2) suggested that the increase in lipid was due to TAG rather than PL, but these data were derived from measures of lipid class composition by TLC and the total lipid data, and so are not independent. Also the data for carbon and nitrogen content were derived from elemental analy- ses and the dry mass data (see Materials and Meth- ods), and so these data too are linked. However the C/N/dry mass and lipid/protein data sets are indepen- dent. This apparent increase in nutrient content was small, and given the experimental error (much of it associated with difficulties of estimating individual egg size) not statistically significant. However the C/N dry mass and lipid/protein data sets are indepen- dent of each other, and since the increase was suggested by two independent measures, it may be real.

An increase in lipid content during the early stages of embryonic development of crustaceans has been demonstrated for a number of polar species (Clarke, 1977, 1983; Clarke et al., 1985), but so far not for a crustacean with a small egg and short development time. The data are equivocal, but suggest that further work would be valuable.

Energetics o f development

The data on proximate composition allow an esti- mate to be made of the energetics of embryonic development. These calculations are complicated by the possibility of the initial increase in lipid content (and hence energy content), and so energetic esti- mates will be limited to the period from day 5 to day 16.

During this period the developing embryo utilized 5.3/~g lipid and 4.8/~g protein (data based on mean composition data in Table 2). The oxygen required for this may be calculated from the standard coefficients (Schmidt-Nielsen, 1979), and its totals 15.2 #g over 11 days; this is equivalent to a respira- tion rate of 1.98/tg egg- ~ day- l, or 82.3 ng egg- ~ hr 1 (5.1ng at O egg ~ hr-l) . If all the nitrogen in the utilized protein was excreted, 4.8/~g protein would release 0.77 #g N (assuming the protein con- tained 16% N). The measured decrease in egg N content over the same period was 0.89#g. Since the composition data suggested that yolk protein was relatively low in nitrogen, the decrease in egg total N content indicates loss of non-protein N (possibly free amino acid). Nitrogen excretion from day 5 to day 16 thus averaged 80.9 ng egg -1 day- 1 or 3.4 ng egg- 1 hr- ~ (0.24 ng at N egg- I hr- l). These data have been averaged over the final 11 days of development. The changes in proximate composition (Table 2) and the patterns of lipid utilisation (Fig. 2, 3) indicate that metabolism will be lower in the earlier stages and higher closer to hatching.

CONCLUSIONS

We may therefore answer the questions outlined in the Introduction as follows:

1. The major fuels for embryonic development are firstly lipid and secondly protein.

2. Both TAG and PL are utilized. All classes of fatty acid are utilized although the major fuels are 16:0 and 18:l.

3. There is only equivocal evidence of an increase in egg lipid during the early stages of embryonic development.

Acknowledgements--We are grateful to Mr W. Hamilton for his care of the broodstock prawns. The prawn research at the Institute of Aquaculture is funded by the Overseas Development Administration (Natural Resources Grants R3874 and R4221) and additional financial help has been received from the European Community under the Science and Technology for Development Programme [Grants TSD A 200 UK (H) and TS2 0099 M (H)]. This support for the work is gratefully acknowledged. The British Antarctic Survey is a component body of the Natural Environment Research Council.

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