Mar Biol (2010) 157:1713–1724 DOI 10.1007/s00227-010-1445-1
ORIGINAL PAPER
The ecological significance of lipid/fatty acid synthesis in developing eggs and newly hatched larvae of Pacific cod (Gadus macrocephalus) Benjamin J. Laurel • Louise A. Copeman • Thomas P. Hurst • Christopher C. Parrish
Received: 29 September 2009 / Accepted: 5 April 2010 / Published online: 24 April 2010 Ó US Government 2010
Abstract The lipid/fatty acid composition of marine fish eggs and larvae is linked with buoyancy regulation, but our understanding of such processes is largely restricted to species with pelagic eggs. In this study, we examined developmental changes in the lipid/fatty acids of eggs and embryos of Pacific cod (Gadus macrocephalus), a species that spawns demersal eggs along coastal shelf edges, but as larvae must make a rapid transition to the upper reaches of the water column. Adult Pacific cod were collected in the Gulf of Alaska during the spawning season and eggs of two females were artificially fertilized with sperm from three males for each female. The eggs were subsequently reared in the laboratory to determine (1) how lipids/fatty acids were catabolized during egg and larval development, and (2) whether lipid/fatty acid catabolism had measurable effects on egg/embryo density. Eggs incubated at 4°C began hatching after 3-weeks and continued to hatch over a 10-day period, during which there was a distinct shift in lipid classes (phospholipids (PL), triacyglycerols (TAG), and sterols (ST)) and essential fatty acids (EFAs: 22:6n-3 (DHA), 20:5n-3 (EPA), and 20:4n-6 (AA)). In the egg stage, total lipid content steadily decreased during the first 60% of development, but just prior to hatch we observed an unexpected 2–3-fold lipid increase (*6–9 lg individual-1) and
Communicated by U. Sommer. B. J. Laurel (&) L. A. Copeman T. P. Hurst Fisheries Behavioral Ecology Program, Alaska Fisheries Science Center, National Marine Fisheries Service, NOAA, Hatfield Marine Science Center, Newport, OR 97365, USA e-mail:
[email protected] C. C. Parrish Ocean Sciences Centre, Memorial University of Newfoundland, Logy Bay, NL A1C 5S7, Canada
a significant drop in egg density. The increase in lipids was largely driven by PL, with evidence of long-chained fatty acid synthesis. Late-hatching larvae had progressively decreasing lipid and fatty acid reserves, suggesting a shift from lipogenesis to lipid catabolism with continued larval development. Egg density measures suggest that lipid/fatty acid composition is linked to buoyancy regulation as larvae shift from a demersal to a pelagic existence following hatch. The biochemical pathway by which Pacific cod are apparently able to synthesize EFAs is unknown, therefore representing a remarkable finding meriting further investigation.
Introduction Understanding the proximal composition of marine fish eggs has been important for bioenergetic modeling (Ronnestad and Fyhn 1993), modeling vertical distribution (Ferron and Leggett 1994), and predicting the nutritional requirements of first-feeding larvae (Sargent et al. 1989). Lipids and fatty acids (FAs) have received particular attention given their critical role in successful early development of marine fish, both as a catabolic substrate and as structural components of cellular membranes (Sargent et al. 1989; Copeman et al. 2002; Tocher 2003). In cold-water, marine fish species with eggs lacking oil globules, yolk lipids are primarily composed of phospholipids (PL) that are rich in (n-3) polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (22:6n-3, DHA, Wiegand 1996). These PUFAs are readily incorporated into the tissue PL of cold-adapted marine species for the maintenance of cell-membrane fluidity at low temperatures (Cossins and Lee 1985; Cossins et al. 1997). They have additional importance as catabolic substrates during early development, with some species catabolizing [50% of
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their polar lipid reserves from egg fertilization to hatch (Tocher 2003). To date, our understanding of lipid metabolism in developing cold-water marine fish eggs/larvae stems from species with pelagic eggs (Sargent et al. 1989). Based on these studies, the dynamics of lipid use has been typified by a pattern in which lipid content in eggs steadily decreases with development until hatch, after which there is often a more precipitous rate of lipid loss in free-swimming embryos (Tocher 2003). However, in species with demersal or bathypelagic eggs, lipids may actually increase toward hatch (i.e., lipogenesis), followed by a progressive decrease after hatch during the free-swimming embryo phase (Finn et al. 1995a, b; Evans et al. 1998; Ohkubo et al. 2006). The prevalence of this pattern across species with demersal eggs is unknown given the paucity of demersal egg studies. Lipogenesis, when noted, has been in species with demersal or bathypelagic eggs that hatch into pelagic larvae (e.g., Atlantic halibut, Hippoglossus hippoglossus, Zhu et al. 2003) and winter flounder, Psuedopleuronectes americanus (Cetta and Capuzzo 1982). For this reason, PL and their accompanying long-chained EFAs may have additional importance in buoyancy regulation, especially for larvae making the rapid transition from deep water to the more highly productive surface waters (Evans et al. 1998; Zhu et al. 2003). In this study, we examine the changing lipid/FA composition of developing eggs and prefeeding larvae of Pacific cod (Gadus macrocephalus). Despite their importance both as predators and as prey in the trophodynamics of the North Pacific (Hunt et al. 2002), very little is known about the early history of Pacific cod, particularly during the egg and larval phase. The transition from a demersal to a planktonic stage is hypothesized to be rapid, but the mechanism by which this occurs has not been examined. We examined changes in the lipid classes and FAs of developing Pacific cod embryos to ask the following questions: (1) Do Pacific cod eggs undergo lipogenesis and depart from our traditional understanding of lipid catabolism during development based on pelagic eggs? (2) Are certain lipid classes and FAs preferentially catabolized (or synthesized) during embryogenesis? and (3) Do these changes in lipid/FA composition correspond with developmental changes in the specific gravity of eggs?
Materials and methods Egg collection Adult Pacific cod were collected by commercial jigging vessels in Chiniak Bay, Kodiak, AK (57°400 N, 152°300 W) in 25–40 m depth during the spawning season in March 2006 and April 2008. Fish were brought aboard the vessel
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alive and determined to be in spawning condition if milt or hydrated eggs were released on the deck. In each collection year, the gametes of ripe males (n = 3) and a female (n = 1) were combined into clean, dry containers (i.e., batches) for a 1-min period before the addition of ambient surface seawater. Seawater was repeatedly added and decanted from egg batches to clean them from excess milt and tissue before transport to shore 4–6 h later. Fertilized egg batches were held for a 2-day period at 4°C at the Kodiak Fisheries Research Center. The egg batch determined to be of highest quality (i.e., round, developing, and [99% fertilization success) was divided into multiple insulated containers (10 ml eggs L-1; *7,000 eggs) and shipped to the Fisheries Behavioral Ecology Program’s experimental laboratory at the Hatfield Marine Science Center in Newport, OR. Parental sizes for the two batches were similar (2006—1 female (72 cm TL), 3 males (62– 68 cm TL); 2008—1 female (68 cm TL), 3 males (60– 68 cm TL)). In the laboratory, eggs were divided into multiple flow-through egg baskets consisting of a 4-L plastic tray with 220 lm mesh sides and solid bottoms. Eggs were scattered in a thin layer covering the bottom of each egg basket at a density of *14,000 eggs/basket in 2006 and *7,000 eggs/basket in 2008. Temperature controlled seawater (4°C) was supplied to egg baskets at a rate of 150 ml min-1 and an air-stone was placed in each tray to increase water flow over eggs during the incubation period. Experimental design In 2006, Pacific cod eggs were sampled from replicate egg baskets (n = 3) at 3 days-post-fertilization (dpf) and every 5–6 days thereafter until the beginning of hatch. At the start of hatch (20 dpf), newly hatched larvae were sampled on 20, 23, 26, and 29 dpf. Remaining larvae in the egg baskets were removed after each sampling period to ensure that only newly hatched larvae were collected in the subsequent sampling period. By restricting our sampling to only newly hatched larvae we were assured that lipid/FA dynamics were based on endogenous energetic resources. Sampling involved pipetting eggs or larvae from an egg tray into a petri dish and sorting under a dissection scope. Viable eggs were apparent in that they were transparent and the embryo inside was in a more advanced developmental stage than the previous sampling period. Samples of 100 eggs/larvae were sorted in this manner, transferred by forceps to a 47 mm ashed glass fiber filter (Whatman GF C), and rinsed with filtered seawater. Excess water was removed by gentle vacuum and samples were transferred to test tubes containing 2 ml of chloroform, flushed with nitrogen, and placed into a freezer at -80°C for extraction and lipid/FA analysis.
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Egg/larval mass measurements were conducted on separate individuals on the same days as the lipid/FA analysis. As much as 20 to 25 eggs or larvae were pooled for dry mass (DM) estimates. Embryos for DM measure were first collectively rinsed in 3% ammonium formate solution to remove excess salt and inorganic material before being transferred to 1.5-cm2 preweighed aluminum foils and an oven set at 68°C. Foils were then stored in a desiccator and weighed. Ash weights were determined by combusting samples in a 450°C oven for 12 h and then re-weighing on a microbalance (Sartorius R16OP) to the nearest 1 9 10-6 g. The total organic content was calculated by using the ash free dry mass (AFDM) and dividing by the number of individuals. The atypical patterns observed of lipid/FA catabolism observed in Experiment 1 (See ‘‘Results’’) prompted a 2nd experiment in 2008. The goals of Experiment 2 were to: (1) determine if patterns were repeatable for another female, (2) increase the sampling resolution to better capture dynamic patterns of lipid/FA catabolism and lipogenesis prior to hatch, (3) reduce egg mortality by lowering the egg density in rearing baskets from 20 ml eggs/basket to 10 ml eggs/basket, and (4) address possible issues of variance in the lipid/FA analysis. The latter was accomplished by both increasing the number of egg baskets from 3 to 4 (thereby increasing the number of samples (n = 4) taken during each sampling period), and adding an internal standard to all lipid samples prior to extraction (see below). With the exception of egg densities in rearing baskets, all rearing conditions were identical between Experiments 1 and 2. Lipid and fatty acid analysis Lipid classes were determined using thin layer chromatography with flame ionization detection (TLC/FID) with a MARK V Iatroscan (Iatron Laboratories, Tokyo, Japan) as described by Parrish (1987). Extracts were spotted on silica gel coated Chromarods and a three stage development system was used to separate lipid classes. The first separation consisted of 20-min developments in 99:1:0.05 hexane:diethyl ether:formic acid. The second separation consisted of a 40-min development in 80:20:1 hexane:diethyl ether:formic acid. The last separation consisted of 15-min developments in 100% acetone followed by 10-min developments in 5:4:1 chloroform:methanol:water. After each separation, the rods were scanned and the 3 chromatograms were combined using T-data scan software (RSS Inc., Bemis, TN, USA). The signal detected in millivolts was quantified using lipid standards (Sigma, St. Louis, MO, USA). Lipid classes were expressed both in relative (mg g-1 wet weight) and in absolute amounts (lg animal-1).
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In 2006, absolute amounts of FA per larvae were calculated using simple conversion factors described by Budge (1999), in which the glycerol, phosphate, and other functional groups are subtracted from the acyl lipid class mass to obtain the mass of fatty acids per lipid class. On the basis of an average fatty acid chain length in seafood, these conversion factors were *0.47 steryl/wax esters, *0.95 triacyglycerols (TAG), *1.0 free fatty acids (FFA), *0.90 diacylglycerols (DAG), *0.37 acetone mobile polar lipids (AMPL), and *0.72 for phospholipids (PL). In 2008, absolute amounts of FA per larvae were calculated using both the calculation based on FA mass per lipid class and an internal standard. Analysis of samples from 2006 without added tricosanoic acid methyl ester (23:0) revealed that it would be a suitable internal standard. Therefore, prior to the extraction of any sample in 2008 an internal standard of 23:0 was added at an amount that was approximately 10% of the total fatty acids. Within sampling date variation was compared at 4 sampling dates using the two methods. As expected, the coefficient of variation using the conversion calculation was higher (*28%) than that of the internal standard (*10%). However, within a given date, mean amounts of fatty acids did not vary significantly between the two methods of calculation. Therefore, we interpreted any variation between the 2006 and 2008 samples to be due to biological variation (e.g., maternal effects) rather than methodological differences in the FA calculation. Total lipid extracts were transesterified using 14% BF 3MeOH for 1.5 h at 85°C. This method derivatized 84– 94% of the acyl lipids. The FAMEs were analyzed on a HP 6890 GC FID equipped with a 7683 autosampler. The GC column was a ZB wax? (Phenomenex, USA). The column length was 30 m with an internal diameter of 0.25 lm and had a 1 m guard column on the front end. The column began at 65°C and held this temperature for 0.5 min. The temperature then ramped to 195°C at a rate of 40°C min-1, held for 15 min, then ramped to a final temperature of 220°C at a rate of 2°C min-1. This final temperature was held for 3.25 min. The carrier gas was hydrogen and flowed at a rate of 2 ml min-1. The injector temperature started at 150°C and ramped to a final temperature of 250°C at a rate of 200°C min-1. The detector temperature stayed constant at 260°C. Peaks were identified using retention times from standards purchased from Supelco (37 component FAME, BAME, PUFA 1, PUFA 3). Chromatograms were integrated using the HP ChemStation Chromatography Software (Version B00.00). Egg density measurements Changing egg density of Pacific cod eggs was determined by measuring sinking velocity multiple times (7, 14, 19,
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and 21 dpf) over the course of the developmental period. Only viable eggs exhibiting normal development and not exhibiting signs of surface fouling were tested. Subsamples of 7 to 20 eggs were pipetted from incubation trays and placed into 50-mL beakers for 30 min of acclimation to one of three salinity treatments. Salinity treatments of 25.3, 31.5, and 37.8 (corresponding to target densities of 1.020, 1.025, and 1.030 g ml-1) were chosen to reflect a diverse range of salinities that eggs might experience from coastal or offshore spawning. Ambient seawater was adjusted to the experimental salinities by addition of distilled freshwater or a commercial sea salt mixture. Following acclimation, individual eggs were pipetted from the acclimation beakers and transferred to the sinking column (1,000-mL glass graduated cylinder). To minimize the effect of injection on sinking rates, a drop of water containing the egg was touched to the water surface, allowing the egg to sink. The time required for the egg to sink a distance of 32.5 cm was recoded. Water temperatures were maintained at 4°C during acclimation and testing. Stoke’s Law was used to convert sinking rates (Ve in cm s-1) of eggs to egg density (qe in g ml-1) according to the equation: qe = (18 lVe) 9 (gD2)-1 ? qw, where l is the kinetic viscosity of the seawater, g is acceleration due to gravity, D is the egg diameter, and qw is the density of the seawater. Kinetic viscosities (in g m-1 s-1) of the three salinities were interpolated from tabled values in Riley and Skirrow (1975). Egg diameter was assumed to be 0.11 cm for all treatments, based on the average egg size observed in measured subsamples. Data analysis Trends in the abundance of lipids and fatty acids were analyzed separately in the egg and larval stage. Data were fit with linear regression models to determine (1) relationships of absolute amounts of lipids or FAs with time post fertilization and (2) catabolism rates of different lipid classes and individual fatty acids with time. In instances, where linear regression did not fit data, non-linear models of up to 3 parameters were tested. Non-linear models were only fit to 2008 data due to insufficient temporal sampling resolution in 2006. Model fits were only presented if a significant relationship was detected; i.e., p \ 0.05. Sinking velocities and egg densities were analyzed with 2-way ANOVA with ‘dpf’ and ‘salinity’ as independent treatment factors. All residuals for single parameter models were inspected for normality. Small departures in normality in the data (e.g., tail curvatures in Nscore plots) were assumed to not influence the overall interpretation of data since sample sizes were equal among comparisons.
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Results Lipids/Fatty acids Both the 2006 and 2008 batches of eggs developed at similar rates. The beginning of hatch occurred at 20 and 23 dpf in 2006 and 2008, respectively. Larvae hatched over a 10–14-day period in both batches. Lipids and fatty acids decreased steadily within the first 60% of development, but subsequently showed a rapid 2–3-fold increase just prior to hatch (Figs. 1, 2). This relationship was ‘u-shaped’ and best described by an exponential decay, exponential linear combination (f = y0 ? ae(-bx) ? cx). The developmental changes of egg/larval lipid content were consistent in both batches although in 2006 the increase in lipid/FA is not seen until the early hatch stage, likely as a consequence of too long an interval between sampling dates (Figs. 1, 2). The higher sampling frequency in 2008 confirms that the increase in lipid and FAs occurs within the egg stage (Fig. 1), including the n-3 and n-6 essential fatty acids (Fig. 2). The large changes in lipid, both during early catabolism and at lipogenesis, were mostly driven by changes in the PL class. This was not surprising given newly fertilized eggs contained *85% PL and subsequently lost 60–80% of their total lipid reserves by the midpoint of the incubation period. The 2006 batch had lower amounts of total lipid compared to the 2008 batch (4.5 vs. 9.7 lg.indiv-1, Table 1), although this may be a consequence of sampling eggs 2 days closer to fertilization in 2008. Compared to Atlantic cod eggs, Pacific cod eggs had a smaller amount of total lipid; however, the relative amounts of fatty acids were very similar (Table 1). The major FA in eggs were DHA * 23 to 28%, EPA * 16%, 18:ln-9 * 10–13%, and 16:0 * 19–22% for both species. Larvae of Pacific cod had notably less DHA and higher amounts of MUFAs than Atlantic cod, but the relative composition of other FAs in larvae appeared to be similar. Differences in lipid class composition were more apparent. Pacific cod eggs had very small proportions of TAG (1–2%) compared to Atlantic cod (*20%) and less than half the percentage of ST (Table 1). Pacific cod lipids were almost entirely comprised of PL (*74–84%), both as eggs and as newly hatched larvae. PL content overall was *50–60% higher compared to Atlantic cod (Table 1). The first larvae to hatch had the highest amounts of total lipid at any point during the sampling period i.e., 7– 10 lg indiv.-1. Larvae hatching at later dates appeared to progressively catabolize lipid during the late incubation stage (Fig. 1). The significant negative regression with time (dpf) indicated catabolism of PL and TAG during this period, whereas a non-significant trend in ST indicated this lipid class was conserved or was possibly increasing
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and MUFAs were more subtle, but still detectable by a 3-parameter model in the egg stage (f = y0 ? ae(-bx) ? cx) and linear regression with dpf for newly hatched larvae. Overall content of FA also mirrored lipid content in that they tended to be higher in the 2008 egg batch. Relative changes in individual FAs differed across the incubation period. Prior to the first hatching, eggs appeared to conserve 16:ln-7 and 18:0 (Fig. 3) and catabolize 20:5n-3 and 22:6n-3 (Fig. 4), although the latter was only seen in the 2008 egg batch and these FAs rapidly increased again just prior to hatch. During late incubation, after hatching had begun, relative decreases were observed in 14:0, 16:ln-7, 18:ln-9, 18:ln-7, and 20:5n-3, whereas increases were observed in 18:0, 20:4n-6, 22:5n-3, and 22:6n-3 (Figs. 3, 4). Figure 5 shows a dramatic increase in DHA:EPA ratios with hatching date, indicating that DHA was becoming increasingly conserved as the incubation time was extended for late-hatching larvae. Proteins
Fig. 1 Changes in the absolute amounts of total lipid, TAG, sterol, and phospholipid in developing eggs and larvae of Pacific cod (Gadus macrocephalus). Values are based on pooled samples of eggs/larvae (n = 100), but expressed as lipid mass (lg) per individual egg or larva. Dotted (2006) and solid lines (2008) represent significant linear or 3-parameter model fits (p \ 0.05; see ‘‘Methods’’) for eggs collected in 2006 (n = 1 female; n = 3 males) and 2008 (n = 1 female; n = 3 males), respectively. Egg and larval periods are plotted separately, but trend lines are not shown if p-values from model fits were [0.05. Larval data represent newly hatched larvae over an extended hatch period of 8–10 days
(Fig. 1). These patterns were consistent both in the 2006 and in the 2008 egg batches although 2008 eggs appeared to have higher amounts of lipid overall. The changes in absolute amounts of fatty acids reflected trends seen in total lipid (Fig. 2). The majority of catabolism was seen in the n-3 PUFAs, followed by a rapid increase in PUFAs just before hatch. Changes in the SFAs
The ash free dry mass (AFDM) and estimated protein and free amino acid (FAA) content of eggs and larvae are shown in Fig. 6. Only 2008 data are shown because sampling resolution in 2006 was too coarse to show dynamic changes in protein during the egg stage. The protein and FAA pool was calculated by subtracting the total lipid mass from the AFDM of eggs/larvae sampled on the same day. Carbohydrates were assumed to be a negligible component of the overall organic content based on other marine fish eggs (Whyte et al. 1993). The changes indicate a slow, steady depletion of protein/FAA followed by a more rapid depletion just prior to hatch. A rapid loss of protein material is observed after hatch due to the loss of the chorion, after which there is a gradual depletion of both lipid and protein/FAA content over the course of the hatch cycle as larvae. Egg sinking rates Pacific cod eggs were negatively buoyant at all salinities tested, with an overall average measured sinking velocity of 0.59 cm s-1. Sinking velocities were significantly affected by both ontogenetic stage (dpf) and acclimation salinity (2-way ANOVA, p \ 0.001; Fig. 7), but not by their interaction (p = 0.754). As expected, due to changes in density of the seawater, egg sinking velocities were significantly higher when acclimated to lower salinities. Egg sinking velocities also declined significantly between 14 and 19 dpf (p \ 0.001) and between 19 and 21 dpf (p \ 0.001). At the intermediate salinity treatment, sinking velocities declined 17% from 0.640 cm s-1 at 7 dpf to 0.530 cm s-1 at 21 dpf.
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Fig. 2 Changes in the absolute amounts of (top panels) short-chained fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) in developing eggs and larvae of Pacific cod (Gadus macrocephalus). Bottom panels show the changes in the absolute amounts of n-3 and n-6 PUFAs with development. Values are based on pooled samples of eggs/larvae (n = 100), but expressed as fatty acid mass (lg) per individual egg or larva.
Dotted (2006) and solid lines (2008) represent significant linear or 3-parameter model fits (p \ 0.05; see ‘‘Methods’’) for eggs collected in 2006 (n = 1 female; n = 3 males) and 2008 (n = 1 female; n = 3 males), respectively. Egg and larval periods are plotted separately, but trend lines are not shown if p-values from model fits were [0.05. Larval data represent newly hatched larvae over an extended hatch period of 8–10 days
Conversion of observed sinking velocities to egg density suggested that egg density at the intermediate salinity decreased from 1.041 g ml-1 at 7 dpf to 1.038 g ml-1 at 21 dpf. Due to osmotic exchange with surrounding seawater, egg density was significantly higher at higher salinities (p \ 0.001).
synthesizing lipids and FAs from other energetic reserves during development. This increase in lipid occurred late in the egg stage and was accompanied by a reduction in egg density shortly before hatch, suggesting that lipogenesis may be important for provisioning buoyant energetic reserves to larvae as they transition from the demersal to pelagic region. Even more surprising were the observed increases in n-3 and n-6 PUFAs during the process of lipogenesis. In vertebrates, n-3 and n-6 PUFAs are considered essential fatty acids (EFAs) that cannot be synthesized de novo. In fact, to date, de novo synthesis of these EFAs (e.g., DHA and EPA) has only been described in bacteria, fungi, and marine algae (Bajpai and Bajpai 1993; Allen and Bartlett 2002). The observed increases in absolute amounts of n-3 and n-6 PUFA, therefore represent a remarkable finding for vertebrates in general via currently unknown mechanisms. Below we discuss these
Discussion Our study is the first to characterize lipid/FA use during embryogenesis for Pacific cod and is one of few to examine these changes in a species with demersal eggs. Although it is generally considered that the lipid and fatty acid composition of fish eggs in oviparous species is limited to those provisioned by the female, from our study it appears that Pacific cod embryos are capable of
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Table 1 Total lipids, lipid classes, and fatty acids in 2-year classes of Pacific cod (Gadus macrocephalus) eggs and newly hatched larvae with reference to literature values for Atlantic cod (Gadus morhua) at similar developmental stages Pacific cod eggs Pacific cod eggs Atlantic cod eggs Pacific cod larvae Pacific cod larvae Atlantic cod (2006, n = 3) (2008, n = 4) (n = 3–4) (2006, n = 3) (2008, n = 4) larvae (n = 3–5) Dry mass per individual (lg)
110.8 ± 2.9
111.1 ± 1.1
116 ± 1.4a a
81 ± 1.4
73.4 ± 1.1
88.1 ± 3.5a
6.6 ± 0.4
10.2 ± 1.3
*11.5a
Total lipid per individual (lg)
4.5 ± 0.2
9.7 ± 1.3
14.8 ± 0.5
Percentage of total lipids TAG
1.0 ± 0.2
1.9 ± 1.3
*20
11.5 ± 1.1
5.7 ± 0.2
4.7 ± 0.7b
FFA
0.6 ± 0.1
3.7 ± 2.1
n/a
\0.1
1.1 ± 0.1
n/a
ST
9.0 ± 0.4
5.4 ± 0.1
*20
12.8 ± 0.7
8.7 ± 1.2
24.6 ± 2.6
PL
83.0 ± 0.8
84.3 ± 1.8
*55
74.4 ± 0.7
77.6 ± 2.9
52.5 ± 4.7
14:0
1.4 ± 0.0
1.6 ± 0.1
1.5 ± 0.8c
0.9 ± 0.0
1.4 ± 0.1
1.6 ± 0.1
16:0
19.3 ± 0.1
Percentage of total FA 22.6 ± 0.2
21.7 ± 0.4
17.2 ± 0.3
20.9 ± 0.2
19.8 ± 0.1
18:0 P SFA
3.3 ± 0.1
2.1 ± 0.1
2.4 ± 0.3
4.2 ± 0.0
4.3 ± 0.2
2.1 ± 0.1
24.9 ± 0.1
28.1 ± 0.1
*25.6
23.1 ± 0.3
28.3 ± 0.1
25.3 ± 0.2
16:1n-7
2.0 ± 1.0
3.8 ± 0.0
4.3 ± 0.1
2.3 ± 0.0
3.7 ± 0.3
2.3 ± 0.1
18:1n-9
13.0 ± 0.2
10.3 ± 0.0
11.8 ± 2.3
10.1 ± 0.0
9.6 ± 0.2
8.1 ± 0.1
18:1n-7 P MUFA
6.6 ± 0.1
5.6 ± 0.0
5.5 ± 0.9
5.8 ± 0.0
5.1 ± 0.1
3.1 ± 0.0
27.2 ± 1.1
23.4 ± 0.2
23.6 ± 1.1
23.0 ± 0.1
22.2 ± 0.2
18.7 ± 0.2
20:4n-6
2.0 ± 0.0
2.1 ± 0.1
3.0 ± 0.3
2.6 ± 0.0
2.3 ± 0.0
1.0 ± 0.0
20:5n-3 22:5n-3
17.4 ± 0.5 1.6 ± 0.0
15.3 ± 0.1 1.5 ± 0.0
15.5 ± 0.7 1.8 ± 0.3
18.4 ± 0.2 1.9 ± 0.0
15.6 ± 0.3 1.6 ± 0.0
16.0 ± 0.2 1.3 ± 0.0
22:6n-3 P PUFA
23.2 ± 0.6
25.7 ± 0.2
27.6 ± 1.7
27.5 ± 0.4
25.5 ± 0.1
32.1 ± 0.1
47.7 ± 1.1
48.1 ± 0.2
46.0 ± 0.3
53.6 ± 0.2
49.0 ± 0.2
56.0 ± 0.3
1.3 ± 0.0
1.7 ± 0.0
*1.8
1.5 ± 0.0
1.6 ± 0.0
2.0 ± 0.0
DHA:EPA Values are mean ± 1 SE a
Finn et al. 1995a, b;
b
Garcia et al. 2008;
c
Salze et al. 2005
results in terms of both their biochemical pathways and ecological/evolutionary significance. Mechanisms of lipogenesis and FA synthesis In general, lipogenesis in fish is rare, but some species are capable of synthesizing lipids using oxidative degradations of amino acids, and all fish are capable of synthesizing SFAs (16:0 and 18:0) via the cytosolic FA synthetase multienzyme complex (Sargent et al. 1989). Further modification of FAs following lipid production is poorly understood, although arguably there should be high selection pressure by fish to elongate or shorten certain FAs to meet physiological demands. All organisms, including fish, are capable of desaturating 16:0 and 18:0 to produce 16:1n-7 and 18:1n-9. FA chain elongation is also relatively common. For example, short-chained PUFAs can be biosynthesized into long-chained PUFAs by fatty acyl desaturase and elongase enzymes (Cook 1996). Alternatively, FA chain elongation can occur under hypoxic conditions via mechanisms described by van Raaij
et al. (1994a, b). In goldfish, van Raaij et al. (1994a, b) demonstrated that amino acids can be acetyl donors for lipogenesis and fatty acid elongation through incorporation of 14C-acetate into FFA, TAG, and PL. Demersal eggs may be more prone to hypoxia than pelagic eggs (Pitcher and Hart 1982). While our culture conditions were set to minimize hypoxic conditions (low egg densities with constant flow rates), it is likely that eggs were supplied with variable amounts of oxygen depending on their proximity to other eggs and location within the incubation tray. More notably, hypoxic conditions can also arise from increased muscular movement within the egg while attempting to tear through the chorion during hatch (Oppen-Berntsen et al. 1990). Despite the aforementioned mechanisms by which Pacific cod embryos are able to increase in lipid content and manipulate FAs shortly before hatch, EFA production (i.e., n-3 and n-6 PUFAs) requires a source of shortchained PUFA precursors. However, these precursors are absent in Pacific cod embryos during the observed increases in PUFAs. To date, there is no described pathway
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Fig. 3 Relative percent change in short-chained and monounsaturated fatty acid composition (14:0, 16:0 18:0, 16:ln-7, 18:ln-7, and 18:ln-9) in developing Pacific cod (Gadus macrocephalus) eggs and larvae. Values are based on pooled samples (n = 100) of eggs or larvae. Dotted (2006) and solid lines (2008) represent significant
linear fits (p \ 0.05), with egg and larval stages plotted separately. Trend lines are not shown if p-values from model fits were [0.05. Larval data represent newly hatched larvae over an extended hatch period of 8–10 days
by which available SFAs (16:0 or 18:0) and MUFAs (16:1n-7 and 18:1n-9) can be elongated to short-chained PUFAs (which can then be further elongated to EFAs). As such, the current view is that EFAs (e.g., EPA and DHA) must come from external food sources or initial provisioning by the mother. The mechanisms and pathways by which Pacific cod synthesize EFAs cannot be determined from this study, and given that protein/FAAs is the likely source of energy for lipogenesis, it is difficult to speculate on the pathways taken since we would not necessarily expect to see decreases in certain fatty acids during the formation or elongation of others. An alternative explanation for the patterns is that we observed extreme cases of selective mortality in each year of the study. However, we believe this to be unlikely, especially since egg mortality was minimized (*\10%) overall in 2008 to address this possible concern. Furthermore, in both years, individual eggs were sorted under a dissection scope to verify that they were alive prior to preparation for lipid/FA analysis. While it is possible that some eggs were on a ‘dead-end’ developmental pathway when they were selected (i.e., were never going to hatch despite developing), it would take a significant amount of
such eggs in the pooled sample of eggs (n = 100) to have a measureable effect in the lipid/FA analysis. Although our sample size was small, with egg batches from only 2 females, the similarity in temporal patterns of lipid content and composition supports our conclusion of lipogenesis during incubation.
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Ecological/evolutionary significance of lipogenesis To our knowledge, our finding that Pacific cod larvae increase their general lipid reserves prior to exogenous feeding is a rare example among fish species. Berg et al. (2001) provided evidence that unfed salmon alevins produced lipid shortly after hatch, but they were not able to rule out the contributions of structural lipids since these were not measured in conjunction with the neutral lipids. Berg et al. (2001) hypothesized that if lipogenesis was indeed occurring in salmonids, it was a reflection of a parent–offspring conflict; i.e., offspring trying to compensate for an under-allocation of resources from the mother who is trying to maximize fecundity. In Pacific cod, we offer another explanation based on similar increases in lipid observed in Atlantic halibut eggs. Evans et al. (1998)
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Fig. 4 Relative percent change in polyunsaturated fatty acid composition (20:4n-6, 22:5n-3, 20:5n-2, and 22:6n-3) in developing Pacific cod (Gadus macrocephalus) eggs and larvae. Values are based on pooled samples (n = 100) of eggs or larvae. Dotted (2006) and solid lines (2008) represent significant linear fits (p \ 0.05), with egg and larval stages plotted separately. Trend lines are not shown if p-values from model fits were [0.05. Larval data represent newly hatched larvae over an extended hatch period of 8–10 days
Fig. 5 Changes in the ratio of 22:6n-3 (DHA) and 20:5n-3 (EPA) of eggs and larvae over time. Values are based on pooled samples of eggs/larvae (n = 100). Dotted (2006) and solid lines (2008) represent significant linear fits (p \ 0.05), with egg and larval stages plotted separately. Larval data represent newly hatched larvae over an extended hatch period of 8–10 days
suggest halibut eggs synthesize lipid to rise out of their bathypelagic spawning region. Winter flounder eggs are also negatively buoyant, but nearly double in total lipid
content at the time of hatching (Cetta and Capuzzo 1982). We propose that Pacific cod are using a similar mechanism to vertically migrate to the upper water column. In larval fish, an increase in buoyancy is often observed concurrent with protein catabolism during the interval between hatching and first feeding (Ferron and Leggett 1994). The combination of the catabolism of ‘‘sinking’’ energetic content (proteins/FAA) to form ‘‘floating’’ energetic content (lipids/FA) at the time shortly before hatch may be a mechanism by which newly hatched larvae are able to ascend to the surface waters and maintain buoyancy there (Riis-Vestergaard 2002). Following hatch, larvae tend to increase their specific gravity up until yolk exhaustion, a trend that is thought to be due to the decrease in lipid stores (Saborido-Rey et al. 2003). At the onset of feeding, buoyancy control is thought to be driven principally by the osmoregulation capabilities of the larvae, a mechanism that is further influenced by the nutritional status of the individual (Frank and McRuer 1989). In Pacific cod larvae, newly hatched larvae are found in the upper reaches of the water column (Brodeur and Rugen 1994; Hurst et al. 2009), so the migration to the water surface would have to be relatively rapid considering the known spawning locations
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Fig. 6 Changes in total lipid, organic and protein/free amino acid (FAA) content of developing eggs and larvae of Pacific cod (Gadus macrocephalus) in 2008. The protein/FAA pool is an estimate based on subtracting the total lipid mass from the AFDM of eggs/larvae sampled on the same day. Carbohydrates were assumed to be a negligible component of the overall organic content based on other marine fish eggs. Values of organic mass are based on means (n = 4) of pooled samples (20–25 individuals) ± 1 s.d. Values of lipid mass are based on means (n = 4) of pooled samples (n = 100 individuals) ± 1 s.d. Dotted line indicates the onset of the hatch period. Data to the right of the dotted line represent newly hatched larvae over an extended hatch period of 8–10 days
Fig. 7 Changes in egg sinking velocities (gray symbols; cm s-1)) and total lipid content (lg indiv.-1) of Pacific cod (Gadus macrocephalus) eggs (black circles) and larvae (white circles) during development. Values for egg sinking rates are based on means (n = 7–20 eggs) at each of 3 water salinities (25.3, 31.5, and 37.8 ppt) per sampling period ± 1 s.d. Values of lipid mass are based on means (n = 4) of pooled samples (n = 100 individuals) ± 1 s.d. Dotted line indicates the onset of the hatch period. Data to the right of the dotted line represent newly hatched larvae over an extended hatch period of 8–10 days
of Pacific cod are at water depths often greater than 100 m in the Bering Sea (Klovach et al. 1995), and likely deeper in the Gulf of Alaska. The rapid biochemical changes
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occurring just prior to hatch and loss of the heavy chorion at hatch may make this a passive process rather than an energetically costly process involving swimming. From an energetics perspective, it would be interesting to analyze the corresponding changes in the FAA pool, the principle energetic component of many pelagic marine eggs (Finn et al. 1995a, b). From previous studies demersal eggs have been considered to have very little energetic reserve comprised of FAA. For example, the FAA component represents 20–50% of the total amino acid (TAA) in pelagic eggs, whereas it represents only *4% in demersal eggs (Ronnestad and Fyhn 1993). However, this conclusion was based on only two marine species, Fundulus heteroclitus, which has atypically large eggs ([2 mm), and Anarhichas lupus, which lacks a larval phase. Based on our estimates, there is a large component of non-lipid organic content in the egg, which is likely FAA and available both as substrate for basal metabolic demands and as the energetic source for lipogenesis prior to hatch. The cost of converting these additional energetic reserves to lipid is unknown, but it is undoubtedly less efficient than catabolizing lipid directly from maternal contributions. In Atlantic halibut, for example, the calories associated with the 18% lipid increase accounted for the majority (73%) of the total calories associated with the decrease of FAA from fertilization to hatching (Zhu et al. 2003). Quantifying the cost of catabolizing one energetic reserve to synthesize another would provide additional insight into the evolutionary significance of these lipid/FA use patterns in developing Pacific cod embryos. The relative changes in FAs showed distinct shifts at the onset of hatch, typical of cold-water marine fish species. DHA:EPA ratios increased markedly at hatch and continued to increase as incubation time increased and larvae hatched later in the hatch cycle. Marine fish larvae generally show preferential conservation of DHA over EPA (Sargent et al. 1999), but it is interesting to see that this ratio is maintained given that the absolute amounts of both fatty acids increased in Pacific cod embryos. On a biochemical level, the DHA:EPA ratio is considered to be the result of competitive interactions between fatty acids for incorporation into phospholipids, specifically competition for the enzymes that esterify fatty acids onto the glycerol backbone (Sargent et al. 1999). Often, this ratio has been explored as a way of predicting or understanding the dietary requirements of marine fish larvae (Tocher 2003; Copeman and Laurel, in review). It is now well-understood that DHA:EPA requirements for marine larvae are speciesspecific, and developmental or dietary departures from the preferred DHA:EPA ratio can negatively impact behavior, growth, and survival of larvae and juveniles (Bell et al. 1995; Copeman et al. 2002; Garcia et al. 2008).
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Conclusions As predicted, PL are the main lipid constituent of Pacific cod eggs and they, along with common PL-bound fatty acids, were responsible for the gross patterns in lipid/FA use observed throughout development. However, the overall changes in lipid followed an atypical pattern of lipid use, with evidence of lipid catabolism shortly after fertilization, followed by rapid lipogenesis in the late egg stage, and finally a steady decrease in lipid during the hatch period. The period of lipogenesis prior to hatch corresponds with significant reductions in the specific gravity of the eggs, suggesting that protein/FAAs are being catabolized to produce lipid, possibly as a means of providing larvae with buoyant, endogenous reserves for the demersal to planktonic transition. During the process of lipogenesis, we observed increased ‘essential’ fatty acid content in eggs and larvae; e.g., DHA and EPA. Given that no such pathways are presently described for de novo synthesis of these fatty acids in vertebrates, this represents a remarkable finding meriting further investigation. Acknowledgments This project was supported in part with funding from the North Pacific Research Board (NPRB) grant #R0605. We thank Drs. Allan Stoner and Michael Davis for reviewing earlier drafts of this manuscript. Thanks also to Scott Haines, Paul Iseri and Michele Ottmar for providing assistance in the laboratory. Brian Knoth and Alisa Abookire assisted with egg collections in the field. Boat charters were kindly provided by Tim Tripp aboard the F/V Miss O. Thanks finally to J. Wells for the patient assistance and laboratory analysis of lipid classes and fatty acids. This manuscript is NPRB Publication # 245.
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