Seasonal and growth stage changes in lipid and fatty acid composition in the multigeneration copepod...

Antarctic Science 23(1), 3–17 (2011) & Antarctic Science Ltd 2010 doi:10.1017/S0954102010000519

Seasonal and growth stage changes in lipid and fatty acidcomposition in the multigeneration copepod Drepanopus

pectinatus from Iles KerguelenP. MAYZAUD, S. LACOMBRE and M. BOUTOUTE

UPMC Universite Pierre et Marie Curie-Paris 6, UMR 7093 CNRS, Laboratoire d’oceanographie de Villefranche,

06230 Villefranche-sur-Mer, France

[email protected]

Abstract: Changes in adult weight, lipid and fatty acid composition per lipid class were studied over a complete

seasonal cycle in Drepanopus pectinatus Brady. This copepod displayed a strong seasonality in adult individual

weight and total lipid content. Stages C3 to adult from a summer cohort were compared in terms of lipid and

fatty acid structure. Changes in lipid content were related to wax esters and polar lipids (PL) while

triacylglycerols (TAG) and cholesterol remained minor constituents except at the end of winter. Changes in fatty

acid composition of different lipid classes showed that food limitation in winter was high enough to affect

22:6n-3 (DHA) content in PL, and provoke accumulation of maximal percentages of bacterial marker as

branched acids. Composition of TAG followed closely the changes in trophic interactions as diatom and

flagellate markers dominated in spring and summer while bacterial and detritus particle markers dominated in

late winter. The composition of wax esters followed the same seasonal succession but with a lower level of turn

over time and shifted characteristics (bacterial markers) from winter to spring individuals when compared to

TAG. Ontogenetic changes showed high triacylglycerols content in younger C3 stages and increasing wax esters

with increasing developmental stages. Fatty acid composition of PL showed low percentages of EPA and DHA

in stage C3, and recovery of high levels of DHA for stage C4 or 20:5n-3 (EPA) at stage C5. The same trend was

observed for 18:4n-3. The reverse pattern was noted for the n-6 polyunsaturated acids (PUFA) suggesting a

greater requirement in younger stages. Fatty acid partitioning between neutral and PL suggested essential fatty

acids selective incorporation from neutral classes into membrane lipids.

Received 20 January 2010, accepted 10 May 2010, first published online 30 July 2010

Key words: energy store, life cycle, lipid dynamics, sub-Antarctic copepod, survival

Introduction

Our knowledge of lipid biochemistry of marine Antarctic

copepods is relatively recent compared to Arctic species and

remains limited to oceanic areas. The earlier studies by

Reinhard & Van Vleet (1986) and Hagen (1988) surveyed the

lipid class composition of the six major species (Calanus

propinquus Brady, Calanoides acutus Giesbrecht, Rhincalanus

gigas Brady, Metridia gerlachei Giesbrecht, Euchaeta

antarctica Giesbrecht and Euchirella rostamagna Wolfenden)

and the fatty acid structure of some of them. More recent work

(see review by Lee et al. 2006) has brought further information

on the triacylglycerol (TAG) and wax ester (WE) structure in

relation to seasons and life cycle variability.

However, to our knowledge, the small neritic species of

both Antarctic and sub-Antarctic areas have received little

attention despite their ecological significance (Tucker &

Burton 1990, Ward & Shreeve 1999). In the Iles Kerguelen,

the zooplankton community is largely dominated by the small

clausocalanid copepod species, Drepanopus pectinatus Brady,

which displays very high spring and summer biomass and

plays a key role in energy transfer towards higher trophic

levels (Razouls & Razouls 1988, 1990, Razouls et al. 1996).

Throughout the year, a succession of four generations with

different duration has been proposed by Razouls & Razouls

(1990), but information on the energetic content and lipid

composition of each generation is totally unknown. As

indicated by Kattner et al. (2007) in their perspectives paper

‘‘there is a general need for comprehensive field studies on

lipid of zooplankton and the seasonal variability of these

changes’’.

During a general survey of the zooplankton community

in Kerguelen, populations of Drepanopus pectinatus were

sampled seasonally in order to examine lipid content,

composition and structure in relation of both food

interactions and life cycle stage.

Material and methods

Zooplankton samples were collected in the Bay of

Morbihan by vertical hauls from bottom to the surface at

central stations (see Fig. 1). Sampling was carried out from

January 1996–February 1997, at monthly or bimonthly

3

intervals depending on seasons for lipid characteristics.

According to Razouls & Razouls (1990) the biological

cycle of Drepanopus pectinatus comprised four generations:

G1 October–December, G2 December–mid February, G3 end

February–mid April, and G4 April–September.

Consecutive temperature and water samples were taken

using reversing temperature probes (Orca RTM T709) at

two depths (0 and 30 m). Samples of 1 litre of surface

water were filtered on GF/F filters for measurements of

particulate chlorophyll a at three depths (0, 30, 80 m) using

a Turner Design 10 fluorometer (Lorenzen & Downs 1966).

Mesozooplankton were sampled with a 200 mm WP2 net by

vertical hauls from bottom to surface. The zooplankton

were immediately diluted in a plastic cooler with surface

seawater and brought back to a laboratory cold room set to

in situ temperature. For seasonal lipid studies, groups of

200–300 C6 females (with few C5 in autumn and winter)

were sorted, immediately deep frozen and kept at -808C

under nitrogen and transported by air shipment to France

on dry ice every 3–4 months. A subsample of female

Drepanopus pectinatus was used for dry weight and total lipid

determination. For dry weight, individuals (10–15) were

placed on aluminium pans and dried at 608C to constant

weight. Weights were measured on a Cahn electrobalance.

On one date in January, D. pectinatus was sorted to

stages to check the influence of ontogeny on the lipid

composition. However, the high number of individuals

needed for complete analyses (800–900 for younger stages,

200 for adults), made it difficult to obtain totally pure

samples. Hence, a subsample of 50 individuals for each

stage was fixed and counted under microscope: group C3

contains 10% of C4 (in number), group C4 contains 5% of

C5, and group C5 contains 3% of females.

Lipid extraction and determination

Lipids were extracted upon arrival in France according to the

method of Bligh & Dyer (1959). After solvent evaporation at

high vacuum, the extracted lipids were weighed in tared vials

on a precision balance ( ± 100 mg) to evaluate the content

of total lipids. The extracts were then placed under nitrogen

at -808C until further analyses, usually within four months.

The very low levels of free fatty acids were taken as evidence

for proper lipid preservation.

Lipid classes were quantified after chromatographic

separation coupled with FID detection on an Iatroscan

Mark V TH 10 (Ackman 1981). Total lipid extracts were

applied to Chromarods SIII using microcapillaries (1 ml)

and analysed in duplicate. Neutral lipids were separated

using a double development procedure with the following

solvent systems: n-hexane: benzene: formic acid 80:20:1

(by volume) followed by n-hexane: diethylether: formic

acid 97:3:1.5 (v/v). Calibration was achieved using either

commercial standards (Sigma) or lipid extract (WE) from

D. pectinatus separated by column chromatography (Mayzaud

et al. 1998).

Fatty acid methyl esters of each lipid class were prepared

with 7% boron trifluoride in methanol (Morrison & Smith

1964). Wax esters fatty alcohols were acetylated using acetic

anhydride (Ackman et al. 1972). Gas liquid chromatography

(GLC) of all esters was carried out on a 30 m length x 0.32 mm

internal diameter quartz capillary column coated with

Fig. 1. Map of sampling sites in

the Bay of Morbihan of the Iles

Kerguelen.

4 P. MAYZAUD et al.

Famewax (Restek) in a Perkin-Elmer XL Autolab gas

chromatograph equipped with a flame ionization detector.

The column was operated isothermally at 1908C for methyl

esters and 2008C for alcohol acetates. Helium was used as

carrier gas at 7 psig. Injector and detector were maintained

at 2508C. Individual components were identified by comparing

retention time data with those obtained from authentic and

laboratory standards. In addition to the examination of esters

as recovered, a part of all ester samples was completely

hydrogenated and the products examined qualitatively and

quantitatively by GLC. The level of accuracy is ± 5% for

major components, 1–9% for intermediate components and

up to ± 30% for minor components.

Data treatment

Non parametric Kolmogorov-Smirnov tests were used to

evaluate significance between mean values using Statgraphics

XV software.

To clarify the seasonal evolution of successive generations

in relation to trophic interactions, the fatty acid profiles of the

wax esters and triacylglycerols were subjected to a factorial

correspondence analysis (Gower 1987). The analysis was

performed on a reduced data matrix transformed to relative

frequencies and scaled so that each row (or column) can be

viewed as a row (or column) of conditional probability

distribution. The fatty acids were used to fill the matrix of

variables and the different sampling dates were used as

observations. Distances between fatty acid profiles were

computed with a x2 metrics. This distance gives symmetry to

the two sets of data (active variables and observations) so that

each factorial axis associated to the cluster of variables

corresponds to a factorial axis of the cluster of observations.

Thus, it was possible to represent simultaneously descriptors

and observations on the plane defined by the factorial axes.

Graphs of projections retained those variables which

displayed more than 1% total contribution to axes 1 and 2.

Hierarchical clustering on the fatty acids descriptors was

Fig. 2. Seasonal changes in sea surface and mid

depth temperature and particulate chlorophyll

at three depths at sampling sites.

LIPID DYNAMICS IN A SUB-ANTARCTIC NERITIC COPEPOD 5

carried out using reciprocal neighbour classification (Lebart

et al. 1995). Computation was made using the SPAD 5.5

software (DECISIA).

Results

Annual changes in environmental conditions

Temperature in the Kerguelen waters followed a very

seasonal pattern with winter lows (July–August) between

2 and 38C and maxima between 6 and 88C from January–

April (summer). Over the period considered the maximum

recorded at both depths were higher in 1997 than in 1996

(Fig. 2 top). Chlorophyll also showed a strong seasonal signal

but with a large interannual variability (Fig. 2 bottom) with a

September spring bloom and succession of summer blooms

in February and March 1996, and blooms of lower intensity in

December, January and February 1997.

Seasonal changes in size, dry weight and lipid content

No attempt was made to redefine the population dynamics and

the scheme with four generations per year was used as

framework of interpretation in the present study. Dry weight

of C5-adult stages showed maximum values in spring and

summer (Fig. 3) following the periods of phytoplankton

abundance. Changes in dry weight were significantly related

to the changes in total lipid content (F1,27 5 12.18,

P 5 0.0017), which displayed similar seasonal pattern with

summer high and winter low concentrations (Fig. 3). These

changes are associated with changes in size, with maximum

value in spring–summer and minimum in winter (Table I).

Seasonal changes in lipid class composition.

The range of total lipid content and dry weight of females

D. pectinatus for each generation are presented in Table I,

together with the corresponding range in composition of

Fig. 3. Seasonal variations of adult female dry

weight and lipid content per individual.

Table I. Ranges in dry weight, size, lipid content and lipid class composition for the different generation (G) of female Drepanopus pectinatus.

G1 G2 1996 G2 1997 G3 G4

spring summer summer autumn winter

Dry weight mg/ind range (min–max) 34.0–91.1 34.4–49.9 46.4–62.0 21.4–26.6 10.5–20.4

Size range (mm/female) 0.96–1.20 1.40–1.70 1.35–1.80 1.28–1.40 0.89–1.20

% dry weight

Total lipid range (min–max) 17.4–78.1 20.9–54.8 10.3–28.1 15.0–44.0 22.6–85.2

% total lipids

Wax esters (min–max) 55.6–86.0 61.6–88.5 61.2–87.6 61.6–85.2 35.8–77.1

Triacylglycerols (min–max) nd–28.2 nd–9.9 nd–18.7 nd 9.1–15.6

Free Fatty acids (min–max) tr–0.6 tr tr tr–0.4 tr–0.8

Phospholipids (min–max) 13.6–35.9 11.5–33.5 12.4–38.2 14.7–38.3 16.2–47.2

nd 5 not determined, tr 5 trace.

6 P. MAYZAUD et al.

lipid classes. WE dominated lipids at all times with mean

percentages ranging from 65 to 73% in winter (G4) and

summer and autumn (G2 and G3) respectively. TAG

showed low contributions to the lipid pool with percentages

ranging from 9–16% in winter (G4) and minimum values

(0–10%) in summer (G2). Polar lipids (PL) remained the

second constituent and ranged throughout the seasons from

14% (spring low G1) to 47% (winter high G4) of total

lipids. Cholesterol and free fatty acids were always low

at the limit of detection. The large range in the content of

lipid within generation is indicative of the high degree of

variability. The differences between means within generation

were usually not significant (P . 0.05) suggesting that, in the

present case, generation may not be the proper time scale to

address the seasonal variation of lipid reserves.

Changes in total lipids were linearly correlated to both polar

and reserve lipids (Fig. 4). WE dominated the lipid dynamics

with a highly significant regression (WE 5 -1.96 1 0.75 Liptot;

P 5 0.0001, F1,28 5 199.1). TAG were also significantly related

to total lipids but with a lower slope (TG 5 -2.19 1 0.21 Liptot;

P 5 0.0004, F1,14 5 22.99). Contrary to the expected trend, PL

lipids were significantly related to total lipids with a slope

similar to that of triacylglycerols (PL 5 3.89 1 0.10 Liptot;

P 5 0.0001, F1,28 5 20.63).

Fig. 4. Relation between total lipids and

concentration of the main lipid classes for

adult females.

Fig. 5. Seasonal variability in lipid classes as

percentage of total lipids for adult females.


Seasonal changes in the main three lipid classes as

percent total lipids (Fig. 5) illustrated the inverse pattern

between PL and WE with high WE percentages in spring

and summer and decreasing levels through winter. TAG

remained low without any clear seasonal pattern. Interestingly,

there was no relation between the catabolism of neutral

lipids and the peak of reproduction between November and

March.

Table II. Drepanopus pectinatus. Dry weight (mg/ind) and lipid class composition (% total lipids per growth stage). WE 5 wax esters,

TAG 5 triacylglycerols, FFA 5 free fatty acids, Chol 5 cholesterol, PL 5 phospholipids.

Stages from G2 generation Dry wt WE TAG FFA Chol PL

C3 1 10% C4 4.1 ± 0.6 25.6 28.9 2.2 nd 43.3

C4 1 5% C5 9.4 ± 2.4 74.9 8.1 1.3 2.8 12.9

C5 1 3% females 25.7 ± 1.9 83.3 3.3 0.8 0.5 13.2

Females 51.8 ± 5.9 70.5 3.3 1.4 1.2 19.7

Table III. Drepanopus pectinatus. Fatty acid composition of the wax esters (% of total methyl esters).

1996 1997

07/02 01/05 02/06 13/07 26/08 26/09 19/11 26/11 07/12 29/01

14:0 2.71 2.69 5.74 6.40 1.16 0.77 4.05 3.37 2.54 0.15

15:0 0.03 0.05 0.09 0.18 0.06 0.02 0.06 0.06 0.04 0.02

ISO15:0 0.03 0.04 0.07 0.06 0.02 0.01 0.09 0.04 0.03 0.04

ANT15:0 0.13 0.03 0.08 0.11 0.02 0.09 0.04 0.09 0.02 0.02

ISO16:0 0.01 0.14 0.12 0.14 0.20 0.06 0.02 0.01 0.01 0.01

ISO17:0 0.18 0.21 0.38 0.34 0.15 0.08 0.29 0.00 0.27 0.00

ANT17:0 0.05 0.06 0.09 0.04 0.03 0.04 0.10 0.15 0.01 0.05

16:0 0.26 0.80 0.24 0.12 0.38 0.21 0.42 0.26 0.18 0.34

17:0 0.57 0.41 0.02 0.49 0.14 0.18 0.80 0.50 0.36 0.39

18:0 0.28 0.37 0.14 0.07 0.13 0.09 0.14 0.12 0.13 0.25

24:0 0.48 0.89 0.42 0.18 0.58 0.50 0.48 0.13 0.81 0.42

S saturates 4.78 5.73 7.43 8.25 2.88 2.05 6.52 4.77 4.43 2.11

14:1 0.59 0.28 0.58 0.46 0.22 0.44 2.41 1.47 0.64 2.59

16:1n-7 12.37 9.85 13.68 21.21 21.80 27.05 14.74 13.48 10.01 12.71

16:1n-5 0.19 0.10 0.08 0.10 0.09 0.08 1.43 0.19 0.05 0.08

18:1n-9 38.57 33.65 40.74 44.09 47.17 45.45 36.95 42.40 29.67 37.42

18:1n-7 0.57 0.86 1.64 2.14 0.65 0.74 0.72 0.78 0.81 0.52

18:1n-5 0.19 0.27 0.19 0.23 0.42 0.30 0.47 0.81 0.13 0.04

20:1n-11 0.92 0.84 1.12 1.79 0.79 0.56 0.43 0.83 0.65 0.90

20:1n-9 0.19 0.57 0.36 0.46 0.27 0.22 0.34 0.22 0.20 0.14

22:1n-13111 0.11 0.34 0.19 0.30 0.17 0.13 0.07 0.07 0.11 0.08

22:1n-9 0.09 0.11 0.13 0.17 0.06 0.02 0.08 0.04 0.07 0.07

24:1n-9 0.33 0.36 0.41 0.31 0.16 0.05 0.13 0.25 0.42 0.21

S monoenes 54.25 47.57 59.42 71.71 72.02 75.16 57.99 60.67 42.86 54.88

16:2n-6 0.21 0.21 0.27 0.26 0.08 0.13 0.14 0.15 0.12 0.24

16:2n-4 1.33 0.93 1.07 1.22 1.03 1.57 0.65 0.18 0.52 1.50

18:2n-6 1.78 2.66 2.25 3.67 3.31 2.45 2.91 1.69 2.61 1.10

20:2n-6 0.19 0.27 0.38 0.38 0.27 0.14 0.31 2.11 1.51 0.24

S dienes 3.60 4.19 4.12 5.67 4.88 4.40 4.10 4.17 4.81 3.19

16:3n-6 0.21 0.29 0.66 0.60 0.25 0.10 0.24 0.33 0.47 0.17

16:3n-4 2.15 0.98 1.10 0.48 0.41 0.83 0.86 0.19 0.44 1.63

16:3n-3 0.16 0.37 0.44 0.47 0.50 0.36 0.56 0.46 0.15 0.11

18:3n-6 0.21 0.24 0.22 0.22 0.11 0.08 0.15 0.14 0.22 0.26

18:3n-3 1.97 2.46 2.40 1.77 2.50 1.96 3.71 4.69 5.12 1.26

20:3n-3 0.06 0.15 0.16 0.13 0.12 0.05 0.09 0.42 0.41 0.06

S trienes 4.76 4.48 4.97 3.66 3.89 3.39 5.62 6.22 6.81 3.50

16:4n-3 0.14 0.35 0.18 0.07 0.33 0.06 0.33 3.04 2.41 0.33

16:4n-1 3.45 1.58 0.73 0.19 0.40 1.33 1.40 0.42 1.13 5.73

18:4n-3 12.84 12.11 11.39 2.74 3.44 2.25 15.30 8.54 17.08 8.96

20:4n-6 0.18 0.35 0.50 0.39 0.28 0.11 0.10 0.57 0.84 0.27

20:4n-3 0.49 0.44 0.46 0.52 0.78 0.35 0.36 0.18 0.49 0.36

22:4n-6 0.21 0.35 0.20 0.39 0.13 0.05 0.00 0.52 0.66 0.08

S poly with 4 18.13 15.62 14.08 4.48 5.60 4.36 17.60 13.35 22.86 16.20

20:5n-3 10.00 11.95 5.48 3.60 4.94 3.45 3.38 4.62 11.07 15.85

21:5n-3 0.85 0.94 0.43 0.22 0.36 0.39 0.42 0.21 0.96 0.91

22:5n-3 0.31 1.18 0.28 0.25 0.50 0.26 0.21 0.10 0.42 0.27

S poly with 5 11.16 14.06 6.19 4.07 5.81 4.09 4.01 4.93 12.45 17.02

22:6n-3 3.23 5.89 2.81 1.20 3.89 3.33 3.65 0.75 4.83 2.76

S PUFA 6 2.97 4.37 4.48 5.90 4.41 3.05 3.85 5.51 6.43 2.35

S PUFA 3 30.53 35.98 24.48 11.00 17.40 12.45 28.01 23.02 42.94 30.88

Sn-6/Sn-3 0.10 0.12 0.18 0.54 0.25 0.25 0.14 0.24 0.15 0.08

8 P. MAYZAUD et al.

The influence of ontogeny was monitored on one occasion

in January using groups of dominant growth stages (Table II).

Because of the limited amount of material, no attempt was

made to weigh total lipid to avoid sample degradation. Early

C3 and C4 stages showed little accumulation of wax esters

and relatively high percentages of TAG and PL. Subadult C5

was the stage of WE accumulation with values exceeding

70% of the total lipids. Triacylglycerols fell below 10% and

PL below 15%. Females retained high percentages of wax

esters. Changes in dry weight showed maximum increase

between stage C4 and C5.

Fatty acid and fatty alcohol constituents of wax esters

The fatty acid composition of WE was dominated at all

times by the monoenes (sum ranging from 47–75% of the

total fatty acids) with oleic acid (18:1n-9) as the main

constituent and to a lesser extent palmitoleic acid (16:n-7)

(Table III). Saturated acids were dominated by myristic

acid (14:0) with maximum percentages in early winter.

Palmitic (16:0) and stearic (18:0) acids were generally low

with values , 1% of total fatty acids. PUFA with 5 and 6

double bonds showed a strong seasonal signal with

minimum values for the winter generation and maximum

values for the summer period (Table III). Polyenes with

four double bonds were the dominant polyunsaturated

(PUFA) fraction with 18:4n-3 ranging from less than 3% in

winter and interesting high levels of 10% during spring and

summer. Substantial amount of 20:5n-3 (10–15%) was

recorded during summer while smaller, but significant,

amounts of 16:4n-1 was abundant in summer. Dienoic acids

were essentially 16:2n-4 and 18:2n-6 while trienoic acids

were dominated by 16:3n-4 and 18:3n-3. The changes in

n-3 PUFA showed marked seasonal changes with a

decreasing trend in autumn, minimal values in winter and

increasing percentages throughout spring and summer.

Changes in n-6 PUFA were less pronounced with lower

percentages in January and February. The resulting ratio

ranged from less than 0.1 to 0.5 with maximal values

during the winter months.

The fatty alcohol fraction of the WE were dominated at

all time by C14 and C16 alcohols (Table IV) which

accounted for over 90% of the total alcohols. Minor

constituents included 18:1n-5 alcohol which displayed

maximal values in winter (August–September) and iso-,

anteiso-branched alcohols, which showed a clear seasonal

pattern with minimum values in summer and maxima in

winter. Unsaturated alcohols with two and three double

bonds were occasionally detected but always in percentages

lower than 0.2% (not shown).

Fatty acid composition of TAG

The fatty acid composition of the TAG fraction was

dominated by saturated and monoenoic acids in similar

proportion ranging from 25–42% and from 23–48% for

saturates and monoenes respectively (Table V). Palmitic

(16:0), myristic (14:0) and to a minor extent stearic (18:0)

acids made the bulk of the saturated acids while oleic

(18:1n-9) and palmitoleic (16:1n-7) acids dominated the

monoenoic acids. Among PUFA 18:4n-3, EPA (20:5n-3) and

to a minor extent DHA (22:6n-3) were the main constituents.

Minor PUFA included 16:3n-4, 16:4n-3, 16:4n-1, 18:3n-3

which are considered as diet markers. Over time, 14:0 and

18:0 dominated in winter and early spring (August–early

November) while 16:0 showed an opposite trend. PUFA (n-3)

showed maximum percentage in December (late spring) and

minimum values in winter (August–September). PUFA (n-6)

displayed a similar pattern of changes but with lower intensity

yielding a S PUFA n-6/S PUFA n-3 ratio which varied from

0.05 in summer to 0.25 in winter (Table V). C20 and C22

monoenes failed to show a clear seasonal pattern with higher

percentages between May and July (autumn–early winter) and

very low values for the rest of the survey period.

Table IV. Drepanopus pectinatus. Fatty alcohol composition of wax esters (% of total alcohols).

1996 1997

07/02 01/05 02/06 13/07 26/08 26/09 19/11 26/11 07/12 29/01

14:0 45.05 39.03 39.72 46.05 39.98 42.99 53.90 47.35 47.47 42.57

ISO15:0 0.60 1.24 1.60 2.03 0.98 0.67 0.61 0.90 0.48 0.40

ANT15:0 0.41 0.63 0.65 0.90 0.45 0.30 0.27 0.32 0.24 0.18

15:0 0.51 1.16 1.28 1.15 0.95 0.71 0.44 0.91 1.22 0.51

ISO16:0 0.17 0.42 0.60 0.57 0.44 0.26 0.11 0.11 0.16 0.11

ISO17:0 0.19 0.82 0.30 0.67 0.61 0.24 0.35 0.33 0.11 0.18

ANT17:0 0.22 0.42 0.40 0.41 0.29 0.10 0.12 0.14 0.10 0.01

16:0 47.92 46.73 47.57 40.15 46.54 44.76 39.43 42.73 46.52 51.38

ISO18:0 0.17 0.46 1.17 0.55 0.50 0.24 0.14 0.07 0.17 0.15

18:0 1.13 2.37 2.26 2.64 1.09 0.45 0.45 0.66 0.75 1.24

S saturates 96.57 93.64 96.47 95.86 92.36 90.94 95.99 93.73 97.36 97.24

16:1n-7 0.26 0.51 0.31 0.37 1.28 2.03 1.57 5.05 0.34 0.31

16:1n-5 0.80 0.50 0.34 0.35 0.45 0.51 0.41 0.41 0.24 0.28

18:1n-5 1.11 1.00 0.64 0.78 1.67 1.67 1.01 0.31 0.70 0.70

S monoenes 2.76 4.56 2.51 2.91 7.04 8.08 3.32 6.04 1.78 1.68


Fatty acids of phospholipids (PL)

Phospholipids showed relatively weak seasonal changes with

DHA and to a lesser extent EPA as the major fatty acids

(Table VI). Saturated acids were the second largest contributor

with palmitic acid as the major constituent (. 22% total fatty

acids) associated with lower levels of myristic and stearic

acids. Monoenoic acids were always intermediate with

percentages , 11% and oleic acid (18:1n-9) as the main

component. Other PUFA included 18:4n-3 and minor

percentages of other n-3 acids (Table VI). The only

seasonal pattern concerned DHA concentration which was

minimal in winter and maximum in spring and EPA and

palmitic acid which showed a reverse pattern of changes.

Table V. Drepanopus pectinatus. Fatty acid composition of triacylglycerols (% total fatty acids).

1996 1997

07/02 01/05 02/06 13/07 26/08 26/09 19/11 26/11 07/12 29/01

14:0 9.13 4.11 1.83 1.49 19.25 15.65 3.13 3.42 5.04 5.65

ISO15:0 0.29 0.21 0.39 0.38 0.46 0.42 0.20 0.13 0.19 0.14

ANT15:0 0.23 0.12 0.25 0.21 0.18 0.24 0.08 0.12 0.12 0.09

15:0 0.22 0.66 0.45 0.51 0.47 0.37 0.24 0.33 0.32 0.16

ISO16:0 1.01 0.20 1.19 0.96 0.17 0.19 0.30 0.76 0.44 0.22

ISO17:0 0.40 0.66 0.80 0.75 0.31 0.33 0.20 0.22 0.49 0.28

16:0 19.11 18.50 20.09 14.51 20.17 19.12 26.36 21.91 13.53 20.58

17:0 1.06 1.48 0.55 1.11 0.21 0.86 0.74 1.49 0.17 0.14

18:0 6.36 5.33 4.12 3.03 0.93 0.82 5.42 6.49 3.30 4.91

20:0 0.34 0.67 0.26 0.48 0.07 0.07 0.48 0.45 0.17 0.18

S saturates 38.43 33.39 31.66 25.43 42.72 39.15 38.74 35.76 24.74 33.49

14:1 0.68 0.43 0.17 0.06 0.23 0.22 0.43 0.24 0.38 1.43

16:1n-7 10.71 5.37 7.60 8.55 16.13 16.08 7.40 5.50 6.42 9.65

16:1n-5 0.53 0.40 0.30 0.26 0.27 0.36 0.51 0.33 0.19 0.28

18:1n-9 18.65 7.94 21.11 9.73 25.42 26.21 20.21 17.81 12.02 19.72

18:1n-7 0.64 1.76 3.82 4.56 1.11 1.80 2.19 1.85 0.77 0.57

18:1n-5 0.83 0.99 1.05 1.25 1.18 1.23 2.14 0.91 0.47 0.62

20:1n-11 0.57 0.25 0.61 1.13 0.33 0.32 0.61 0.19 0.27 0.52

20:1n-9 0.14 1.09 2.03 0.76 0.44 0.30 0.15 0.28 0.13 0.19

20:1n-7 0.09 0.72 0.65 1.76 0.19 0.13 0.11 0.05 0.04 0.03

20:1n-5 0.16 0.25 0.17 0.33 0.15 0.11 0.36 0.11 0.13 0.08

22:1n-13111 0.04 6.90 0.45 5.04 0.09 0.13 0.08 0.05 0.07 0.03

22:1n-9 0.11 3.77 0.50 2.96 0.12 0.21 0.20 0.00 0.10 0.08

S monoenes 34.87 32.90 41.43 41.97 46.41 47.99 35.36 28.59 23.17 34.33

16:2n-4 1.10 0.50 0.49 0.61 0.74 0.92 0.22 0.21 0.46 1.16

18:2n-9 0.13 0.25 0.14 0.33 0.05 0.07 0.73 0.23 0.09 0.04

18:2n-6 0.99 2.13 1.66 2.28 1.61 1.26 2.30 1.36 1.71 0.69

18:2n-4 0.16 0.21 0.43 0.36 0.10 0.12 0.05 0.39 0.08 0.43

20:2n-6 0.10 0.25 0.43 0.45 0.18 0.12 0.24 0.99 0.71 0.10

S dienes 2.58 3.56 3.30 4.17 2.70 2.68 3.91 3.32 3.24 2.54

16:3n-4 1.45 0.42 0.29 0.41 0.08 0.23 0.22 0.14 0.38 0.95

16:3n-3 0.19 0.47 0.40 0.42 0.23 0.29 0.47 1.00 0.64 0.06

18:3n-6 0.12 0.15 0.09 0.18 0.02 0.04 0.52 0.60 0.13 0.28

18:3n-3 1.24 1.80 1.51 1.85 1.22 1.14 2.67 5.15 4.45 0.96

20:3n-3 0.09 0.41 0.27 0.17 0.06 0.05 0.09 0.27 0.30 0.06

S trienes 3.42 3.59 3.05 3.40 1.61 1.75 4.11 7.73 6.55 2.44

16:4n-3 1.08 0.61 1.09 0.63 0.06 0.07 0.29 2.14 1.62 0.26

16:4n-1 2.56 0.55 0.43 0.39 0.14 0.37 0.25 0.28 0.07 3.64

18:4n-3 7.81 7.23 4.48 5.26 0.69 1.19 8.04 10.65 17.79 7.42

18:4n-1 0.26 0.31 0.15 0.34 0.13 0.17 0.26 0.38 0.39 0.36

20:4n-6 0.06 0.20 0.27 0.42 0.06 0.10 0.07 0.30 0.39 0.12

20:4n-3 0.24 1.08 0.59 0.48 0.15 0.17 0.25 0.22 0.41 0.27

S poly with 4 12.12 10.06 7.42 7.86 1.45 2.30 9.46 14.30 21.05 12.10

20:5n-3 5.02 7.64 6.65 9.31 2.42 3.65 2.71 5.93 9.88 10.64

21:5n-3 0.56 0.74 0.46 0.68 0.57 0.19 0.48 0.53 1.28 0.79

22:5n-3 0.20 0.93 0.82 1.10 0.22 0.25 0.26 0.28 0.65 0.03

S poly with 5 5.80 9.42 8.00 11.25 3.20 3.86 3.45 6.78 12.21 11.84

22:6n-3 2.47 7.17 5.14 5.93 1.92 2.28 4.98 3.53 9.09 3.26

S n-6 PUFA 1.58 3.01 2.84 3.61 1.88 1.52 3.28 3.51 3.52 1.31

S n-3 PUFA 18.90 28.09 21.42 25.82 7.54 9.27 20.24 29.71 46.11 23.76

Sn-6/Sn-3 0.08 0.11 0.13 0.14 0.25 0.16 0.16 0.12 0.08 0.06

10 P. MAYZAUD et al.

Among the branched acids, iso15:0, iso17:0 and anteiso17:0

displayed a clear winter maximum.

Fatty acid composition of ontogenetic stages

Changes in fatty acid composition from copepodite C3 to

adult stages are presented in Table VII for both total polar

and neutral lipids. Polar lipids exhibited a major decrease

in total saturates and monoenes with growth stage and

conversely increasing percentages in PUFA. Copepodite

C3 displayed the smaller level of DHA and EPA and

maximum values for C5 and adult female. The same

applied at a smaller scale for 18:4n-3. Conversely, palmitic

and stearic acids, and to a minor extent palmitoleic and

oleic acids, were maximum for C3 and decrease sharply as

early as C4 stage. The TAG pattern displayed was different

with increasing percentages of total saturates and dienoic

acids, decreasing levels of total monoenoic acids while

PUFA did not show a clear pattern of variation. Palmitic

acid, palmitoleic and DHA displayed the largest increase in

percentages, while oleic acid, stearic acid, linoleic acid

decrease mainly between C3 and C4 stages. The same

pattern was observed for 18:4n-3 but with a decrease

between C4 and C5. EPA and DHA showed little changes

with no clear pattern. WE showed increasing percentages

of total monoenoic acids and dienoic acids but no clear

pattern for the sum of saturated acids. Conversely, total

PUFA showed decreasing percentages with increasing

copepodite stages. The decrease was essentially associated

with 18:4n-3, DHA and linoleic acid, which dropped either

between C3 and C4 or between C4 and C5 and to a minor

extent with oleic, palmitic and stearic acid. Increasing

percentage concerned essentially 16:4n-1, 16:3n-4, 16:2n-4

and myristic acid.

To illustrate the probable changes in trophic interactions

with growth stage, a factorial correspondence analysis

(FCA) was performed on the fatty acid composition of the

TAG from stage C3 to adults. Due to the limited number

of cases the first two factorial axes accounted for 96% of

the total variance and the link between growth stage and

characteristic fatty acid grouping can be derived from

a hierarchical clustering on factorial score from all three

Table VI. Drepanopus pectinatus. Fatty acid composition of phospholipids (% total fatty acids).

1996 1997

07/02 01/05 02/06 13/07 26/08 26/09 19/11 26/11 07/12 29/01

14:0 1.94 1.22 1.54 1.38 3.06 2.03 1.83 0.94 1.22 0.39

ISO15:0 0.16 0.25 0.30 0.28 0.40 0.13 0.12 0.07 0.09 0.04

ANT15:0 0.07 0.12 0.11 0.10 0.20 0.05 0.04 0.03 0.03 0.02

15:0 0.18 0.36 0.39 0.33 0.71 0.29 0.21 0.14 0.20 0.12

ISO16:0 0.16 0.50 0.22 0.26 0.30 0.16 0.09 0.04 0.12 0.17

ISO17:0 0.28 0.36 0.39 0.39 0.67 0.42 0.28 0.06 0.15 0.23

ANT17:0 0.12 0.13 0.24 0.23 0.31 0.13 0.06 0.06 0.06 0.05

16:0 19.99 14.61 25.38 21.17 19.73 18.27 22.62 21.14 18.81 17.71

17:0 0.23 0.56 0.58 0.51 0.79 0.30 0.26 0.29 0.31 0.22

ISO18:0 0.15 0.12 0.58 0.41 0.24 0.22 0.18 0.10 0.19 0.08

18:0 2.04 2.44 1.77 1.10 2.06 1.22 1.59 1.32 1.27 2.99

S saturates 25.34 20.67 31.50 26.16 28.47 23.22 27.28 24.19 22.45 22.02

16:1n-7 1.09 1.12 1.04 1.21 2.07 2.15 0.35 1.08 0.65 0.78

18:1n-9 2.07 2.78 2.77 2.53 3.75 4.37 1.82 1.79 1.75 2.72

18:1n-7 0.47 0.80 0.98 1.10 2.05 2.34 0.98 1.01 0.51 0.39

18:1n-5 2.86 1.17 2.32 2.49 1.07 1.58 3.91 2.31 2.47 2.27

24:1 0.58 4.43 2.42 1.88 0.74 1.06 1.41 1.81 1.80 2.51

S monoenes 7.08 10.30 9.65 9.35 9.69 11.50 8.48 7.99 7.18 8.67

18:2n-9 0.10 0.09 0.16 0.27 0.23 0.14 0.79 0.42 0.08 0.02

18:2n-6 0.63 1.23 0.58 0.80 0.96 0.79 1.03 0.71 0.55 0.54

18:2n-4 0.12 0.08 0.11 0.12 0.13 0.15 0.05 0.04 0.07 0.13

S dienes 0.85 1.40 0.84 1.19 1.33 1.08 1.87 1.18 0.70 0.69

16:3n-3 0.06 0.16 0.11 0.12 0.14 0.09 0.06 0.04 0.04 0.06

18:3n-3 0.45 0.59 0.39 0.47 0.71 0.47 0.53 0.71 0.55 0.21

S trienes 0.51 0.75 0.50 0.59 0.85 0.56 0.59 0.75 0.59 0.27

16:4n-3 0.06 0.48 0.04 0.03 0.29 0.10 0.06 0.11 0.15 0.28

18:4n-3 3.40 2.13 1.71 1.55 1.56 1.46 2.05 1.94 1.96 2.42

20:4n-3 0.38 0.78 0.27 0.22 1.02 0.64 0.38 0.24 0.27 0.33

S poly with 4 3.84 3.39 2.02 1.80 2.87 2.21 2.49 2.29 2.38 3.03

20:5n-3 19.97 14.52 13.79 18.68 16.70 24.00 16.62 20.35 20.40 22.14

21:5n-3 0.35 0.52 0.24 0.26 0.66 0.70 0.16 0.30 0.30 0.34

22:5n-6 0.15 0.41 0.20 0.26 0.33 0.15 0.08 0.25 0.65 0.17

S poly with 5 20.47 15.45 14.23 19.20 17.69 24.85 16.86 20.90 21.35 22.65

22:6n-3 38.63 43.27 37.85 38.43 32.01 31.77 41.01 40.03 43.29 39.77


axes. The results presented in Fig. 6 showed a strong

opposition on axis 1 (85% total inertia) between stage C3

and older stages with C3 associated with saturated and

branched acids as well as (n-3) 16 PUFA and 18:4n-3. This

suggests feeding on small-size particles (live and detritic).

Later stages showed a clear association with diatom bloom

type material with C16 PUFA of the (n-4) and (n-1)

families, 16:1n-7 and EPA. Interestingly, the second axis

Table VII. Changes in fatty acid composition (% total fatty acids) of the different classes of lipids for copepodite stages and adult females of

Drepanopus pectinatus. PL 5 phospholipids, TG 5 triglycerides, WE 5 wax esters.

PL C3 PL C4 PL C5 PL fem TG C3 TG C4 TG C5 TG Fem WE C3 WE C4 WE C5 WE fem

S Saturates 53.16 29.79 24.66 24.73 31.16 33.06 39.05 35.75 3.65 3.83 4.50 4.90

S Monoenes 22.83 10.20 11.15 9.62 42.35 30.75 34.11 37.05 53.52 54.24 61.63 63.47

S dienes 1.59 0.53 0.56 0.59 2.54 2.86 2.65 3.44 3.89 4.18 4.94 4.89

S poly 3 1.59 0.73 0.66 0.68 4.08 4.09 2.65 3.44 5.06 4.90 4.71 4.63

S poly 4 1.63 2.29 2.08 2.61 8.03 13.23 7.34 7.14 18.03 18.92 11.64 10.42

S poly 5 6.32 18.91 23.61 25.15 7.05 8.05 8.26 7.45 11.49 11.28 9.47 8.36

14:0 - - - - 0.34 5.18 5.80 7.11 0.79 2.57 4.25 4.58

16:0 38.02 24.61 20.89 20.01 15.58 21.18 27.59 23.29 1.15 0.18 0.12 0.15

18:0 10.10 2.45 1.65 1.45 10.57 4.18 2.85 2.17 0.98 0.16 0.11 0.10

16:1n-7 3.12 0.92 1.04 0.92 7.99 7.57 8.24 9.24 8.46 11.20 19.08 19.75

16:1n-5 0.63 0.11 0.08 0.05 - - - - - - - -

18:1n-9 9.78 3.36 4.44 4.03 30.89 16.82 20.28 22.21 43.30 38.07 38.66 39.05

18:1n-7 3.92 0.49 0.45 0.39 - - - - - - - -

18:1n-5 4.20 2.91 2.78 2.44 - - - - - - - -

16:2n-4 0.70 0.07 0.07 0.07 0.29 0.62 0.70 0.83 0.61 1.20 1.90 1.94

18:2n-6 0.94 0.69 0.63 0.64 2.01 1.64 1.31 1.73 2.58 2.41 1.87 1.89

18:2n-4 0.84 0.20 0.10 0.09 0.07 0.14 0.17 0.42 - - - -

16:3n-4 - - - - 0.62 0.43 0.31 0.26 0.48 1.04 1.37 1.35

16:3n-3 - - - - 0.43 0.14 0.19 0.22 - - - -

16:4n-3 - - - - 0.11 0.53 0.53 0.82 - - - -

16:4n-1 - - - - 0.21 1.41 0.87 0.80 1.34 2.49 1.94 1.86

18:4n-3 0.80 1.59 1.42 1.76 7.48 10.62 5.32 4.74 15.54 15.07 7.98 7.20

20:4n-6 0.49 0.19 0.24 0.24 0.04 0.10 0.16 0.23 - - - -

18:5n-3 - - - - 0.79 0.24 0.08 0.07 1.16 0.08 0.03 0.02

20:5n-3 6.03 18.12 22.57 24.14 5.62 6.39 7.44 6.61 9.31 10.15 8.82 7.76

22:6n-3 11.84 34.96 36.17 35.58 4.78 5.77 5.17 5.23 4.57 4.31 1.51 1.18

Fig. 6. Hierarchical cluster analysis on the

score from the factorial correspondence

analysis on triglyceride fatty acids from the

different copepodite stages (C3 to adults).

The clustering illustrates the results of the

FCA considering the projections on the first

three axes and summarizes the associations

between growth stages and fatty acids in all

three directions of the factorial space.


(11% total inertia) discriminate C4 from C5 and adults

suggesting a differential use of the diatom bloom material,

but similar trophic relationships for C5 and adults.

Seasonal changes in fatty acid composition and trophic

interactions of adult females

To clarify the importance of season, FCA was performed

on the fatty acid constituents of the wax esters and

triacylglycerols fractions. For WE, three factorial axes are

needed to explain 75.3% of the total variance (axis 1 5 32.5%,

axis 2 5 26.3%, axis 3 5 16.5%). Projection on the factorial

plan defined by the first two axes opposes the summer period

(January–February) to the spring one (November–December)

on the first axis (Fig. 7 top). Spring and summer individuals

are opposed to the winter ones (July–September) on the

second axis. The third axis (not shown), singles out the 19/11

sampling date. The seasonal path derived from the wax esters

showed a transition from summer to autumn and to winter

along the second axis. An internal shift within wax esters

fatty acids of the winter individuals can be seen along the

first axis from July–September. In terms of fatty acid

descriptors the opposition on the first component between

summer and spring is characterized respectively by 16

PUFA, EPA versus a group of n-3 and n-6 PUFA (16:4n-3,

18:3n-3, 20:3n-3, 20:2n-6). The winter generation on

the second axis is associated with saturates, monoenoic

acids and 16:3n-3. The winter changes suggested that

August–September wax esters showed increasing proportion

of some PUFA (16:2n-4, 20:4n-3).

The projection of the TAG fatty acid confirmed a globally

similar seasonal pattern except that the factorial analysis

suggested a different relationship between variance and

processes involved. Indeed, axis 1 accounted for 41.6% of

Fig. 7. Factorial correspondence analysis on the

seasonal changes in fatty acid composition

for wax esters (top) and triglycerides

(bottom). Top: combine projections of wax

ester fatty acids and sampling dates. Bottom:

single projection of triglycerides sampling

dates. The figure shows the ordination of the

variables and observations in the plane of

axis 1 and axis 2. The major trend of

seasonal variation is illustrated by the dashed

line arrows joining the sampling dates. Fatty

acids illustrate for each season the key

descriptors associated with the time periods.


the total variance and singles out the shift in triglyceride

composition between early and late winter (Fig. 7 bottom).

The second axis opposes the spring–summer individuals to

the winter one but with only 26.8% of the total inertia. The

fatty acid descriptors are relatively similar to the one

recorded for the wax esters with spring associated with (n-3)

16, 18 PUFA and EPA, and summer characterized by (n-1)

(n-4) C16 PUFA and 14:1 (not shown). The triacylglycerols

from winter individuals are divided into two groups: June–July

associated with 22:1 and 20:1 acids and August–September

associated with 14:0, 16:0, C16 and C18 monoenes, and

branched fatty acids.

Discussion

The copepod Drepanopus pectinatus is considered endemic

to Kerguelen and constitutes 70–98% of the total copepod

population (Razouls & Razouls 1988). Based on cohort

analysis, its biological cycle has been shown to comprise

four generations of different durations (44–49 days in summer

to 151 days in winter) which vary in relation to temperature

and food availability, with populations growing from G1 to

G3 and slower development or possibly arrest of moulting for

the winter generation G4 (Razouls & Razouls 1990).

In copepods, lipid accumulation has been classically related

to seasonality in food supply and reproductive metabolism

(see review by Lee et al. 2006). Large accumulations of wax

esters in copepods or euphausiids are usually related to their

overwintering strategy (with or without diapause), while the

link with reproduction is more complex with either species

fuelling their egg production with accumulated lipids or

species using food - derived lipid to cover their reproductive

needs. As observed by Lee et al. (2006) most data at present

concerns species from polar or subpolar regions, i.e. large

Calanus type copepods and little is known concerning the

lipid dynamics of lipid-poor or lipid-rich small neritic

copepods. An earlier report on different small species from

the North Sea, such as Pseudocalanus elongatus, Acartia

clausi and Centropages hamatus, demonstrated that lipid

content was directly related to spring and summer blooms of

phytoplankton (Kattner et al. 1981). Most of these species

have two or more generations per year and the relationship

between lipid dynamic, generation succession, trophic

environment and reproductive strategy is largely unknown,

though it has generally been assumed that egg production was

directly or indirectly related to intake or synthesis from

available food source (Corkett & McLaren 1969, Bautista

et al. 1994).

Our data on Drepanopus pectinatus illustrated that,

although this is a multigenerational species, intra-generation

variability of lipid class composition dominated. Indeed,

spring and summer generations showed large fluctuations

while the winter generation showed a strong seasonal trend

throughout the period. As a result the seasonal lipid dynamics

did not seem to be linked to any single generation but rather to

the general pattern with a strong winter decline. A possible

general explanation could be that the lipid dynamics of a

given generation is driven by the accumulation of the previous

generation of females. Hence, spring and summer variability

in female lipid class composition could be linked to

successive phases of neutral lipid accumulation and

catabolism related to the high reproductive rate taking

place during this period of time (Alonzo et al. 2001). Late

autumn–winter decrease in lipid reserves is probably a

response to both low food conditions and lower level of

reproduction entirely based on the internal pool of lipid

reserves (Alonzo et al. 2001). Although total lipid content

is moderate (30–40% dry weight) when compared to large

Calanus species (Lee et al. 2006), the seasonal dynamics of

lipids was related to the accumulation of wax esters in

spring and summer (reproductive period). The coincidence

between high phytoplankton biomass and high wax esters

content does not mean that lipid accumulation controlled

egg production since Alonzo et al. (2001) established

that D. pectinatus used ingested phytoplankton to reduce

the consumption of their internal lipid reserves. The

seasonal changes in total lipid and WE suggested that for

multigeneration species, the overwintering pattern with high

lipid accumulation does not apply since winter individuals

showed the smallest size and lowest lipid levels. However,

the relatively high proportion of WE in late autumn and the

decreasing trends as winter developed is indicative that

survival remains associated with this type of reserve but

determined by the previous generation of females. The decrease

in WE between June and October could well illustrate the

importance of the long generation time of G4. Early winter

subadults and adults probably originate from the autumn G3

generation with relatively high wax esters percentage, while

individuals found in late winter in August were probably new

recruits from the winter G4 population with low WE but

relatively high TAG percentages.

Fatty acid composition of D. pectinatus cannot be

compared to any similar species from the Southern Ocean,

since data is non-existent. Polar lipid structure showed a

dominance of DHA, EPA and 16:0, a feature common to all

marine planktonic crustaceans (Albers et al. 1996, Mayzaud

1997, Lee et al. 2006). However, the changes in PUFA

proportion with season and generation illustrate the food

limitation on the winter generation to ensure proper

membrane structure. The lower content in DHA and

18:4n-3 together with the relatively stable level of EPA

suggests limitation of DHA synthesis in relation to the very

low level of food supply and the detritic nature of the

particulate matter associated with the maximum of

heterotrophic bacterial abundance (Razouls et al. 1997).

Such a dietary constraint is expected to be even more

influential on the control of the composition of neutral

lipid. Indeed, triacylglycerols changes are indicative of the

origin of food ingested over a relatively short time scale


and follow quite well the changes in particulate characteristics

(Razouls et al. 1997). The spring and summer individuals

were associated with markers of phytoplankton blooms: i.e.

spring females with both diatoms and flagellates markers

(PUFAs of the n-3 family) and summer ones with diatom

(C16 PUFAs). Autumn and early winter individuals appeared

to shift feeding towards a more omnivorous/carnivorous mode

with C20 and C22 monoenes as characteristic descriptors (see

Lee et al. 2006). Late winter individuals are clearly associated

with detritus/bacteria food chain associated with saturated and

branched chain fatty acids. Wax esters illustrated a slightly

different perspective related to the longer time integration of

the fatty acid composition (Lee et al. 2006). Hence, WE from

the spring individuals are related to flagellate and some

bacterial descriptors (18 PUFA, 16:4n-3, 20:3n-3 but also

anteiso17:0, iso 17:0, 16:1n-5), while summer WE are clearly

associated with diatom bloom markers (C16 PUFA and 20:5).

Autumn and winter WE, associated with monoenes (C16, C20

and C22 as well as 16:3n-3, 20:4n-3 and 18:2n-6), would

indicate a more omnivorous/carnivorous trophic mode.

The alcohol fraction is extremely stable in all generations

with mainly saturated constituents accounting for more than

90%. The dominance of 16:0 and 14:0 alcohols is shared with

other neritic copepods such as Pseudocalanus sp. (Fraser et al.

1989), Pseudocalanus acuspes (Norrbin et al. 1990) and

Pseudocalanus minutus (Mayzaud 1980, Lischka & Hagen

2006). Kattner et al. (1996) suggested that such biosynthetic

pathway is associated with omnivorous/carnivorous trophic

behaviour. Given our results and the phytoplankton regime of

late stages D. pectinatus in spring and summer, one could

question this interpretation. Sargent et al. (1976) suggested

that wax esters formation in copepods was a mechanism

which ensures a high rate of lipid synthesis by converting

inhibitory end products fatty acyl coenzyme-A into alcohols,

which are removed from the system by being coupled with

fatty acids of dietary origin. Fatty alcohols may originate

either from phytoplankton fatty acids or synthesized de novo

from carbohydrates or amino acids (Sargent & Henderson

1986). In both cases D. pectinatus limits conversion to short

chain alcohols, in contrast to large Calanus species, which

convert monoenoic acids into 20:1n-9 and 22:1n-11 alcohols.

All large calanoid species, which undergo diapause during

winter to cope with low food conditions, accumulate wax

esters over the short phytoplankton abundance with a

dominance of high energy long chain C20 and C22

monoenoic acids (Falk-Petersen et al. 2009). Small, neritic

calanoid copepods, such as D. pectinatus, also feed mostly

on phytoplankton during spring and summer and display a

high rate of lipid synthesis. The outcome of these reserves

seems unlikely to be oriented towards winter survival since

few individuals from summer generation are likely to survive

as long as winter. None of the small species mentioned are

known to diapause and probably do not require such long

chain high energy molecules for their metabolic processes.

Other species with short chain saturated alcohols include

herbivorous/omnivorous Calanus propinquus (Hagen et al.

1993), Metridia longa (Sargent & Henderson 1986), Euchaeta

japonica (Lee et al. 1974) and carnivorous species such as

Paraeuchaeta antarctica (Mayzaud unpublished data). Hence,

long chain alcohols are more likely to be related to life

cycle requirements associated with diapause than trophic

type.

The changes in lipid classes over growth revealed some

interesting features. Young D. pectinatus copepodites showed

a dominance of TAG at stage C3, which is gradually replaced

by wax esters from stage C4 to a maximum percentage during

stage C5. Comparison is again difficult since most relevant

data concerns large calanoid species but the increased content

of wax esters with copepodite stage is consistent with the

findings of Lee et al. (1974) on Euchaeta japonica, and of

Kattner et al. (1994) on Calanus propinquus, Calanoides

acutus and Rhyncalanus gigas. The decrease in TAG seems

more specific to such small species and may simply indicate a

slow change from triacylglycerols dominating eggs and

nauplii to low triglyceride/high wax esters composition in

late copepodite and adult stages.

Fatty acid changes in relation to ontogenetic changes have

been studied in different copepod species but generally with

regard to total lipid rather than lipid classes or only for late

copepodite stages (Lee et al. 1974, Kattner & Krause 1987,

Kattner et al. 1994, Ward et al. 1996). Hence, comparisons

with the present findings are difficult due to the confounding

effect of neutral lipid changes on the polar lipid composition.

One of the interesting features recorded in the present work is

the low percentages of EPA and DHA in the phospholipids of

copepodite stage C3, and the recovery of high levels of DHA

as early as stage C4 or EPA at stage C5. The same trend was

observed for 18:4n-3 but over a smaller range of percentages.

The reverse pattern could be seen for the n-6 PUFA suggesting

a greater requirement in younger stages. Confirmation of

this possibility requires further study on a more complete

set of developmental stages. Saturated and monoenoic acids

decreased with increasing growth stage.

Considering the limited ability to elongate and/or desaturate

18-carbon PUFA (Moreno et al. 1979), copepods require pre-

formed essential fatty acids such as arachidonis acid (ARA,

20:4n-6), EPA and DHA in their diet to sustain growth.

Although our study was not organized to study essential fatty

acid (EFA) requirements, the data on the fatty acid partitioning

between neutral and PL suggests useful information

concerning EFA incorporation into membrane lipids. The

proportions of DHA, EPA and to a lower extent ARA recorded

in all stages (respectively 12–36%, 6–25% and 0.2–0.5%) were

markedly higher than in the natural spring–summer particulate

matter used as diet by the small copepods (DHA: 0–6%, EPA:

1–10%, ARA: 0.1–2%; Kerguelen shelf water, Mayzaud

unpublished data), indicating selective incorporation into

membrane lipids. This suggests that diet lipids are very

probably limiting in terms of DHA and EPA incorporation

mainly for the early C3 stage which feeds on the smaller


fraction of the size particles with low PUFA content

(Mayzaud et al. 1989).

In conclusion, while yearly and multiyear generation

copepods have developed lipid accumulation to respond

to winter survival and reproduction, multigenerational

species such as Drepanopus appear to accumulate lipid for

immediate specific needs, with spring and summer generation

accumulation to ensure reproduction and autumn generation

accumulation followed by winter catabolism to overcome

late winter poor food conditions. The dominant control of

neutral lipid composition by food intake, illustrated well

the succession of trophic interaction imposed on each

generation and the differential food sources for the different

developmental stages. Assimilated and stored (n-3) as well

as (n-6) PUFA are required to maintain structural synthesis,

but further work on a larger set of developmental stages

would be required to confirm our preliminary results.

Acknowledgements

The study was part of the IOZ programme financially

supported by a grant from the Groupement de Recherche en

Environnement 1069 ‘‘Ecosystemes Polaires et Anthropisation’’

from the CNRS, from IPEV Interactions Oiseaux-

Zooplancton 166 (IOZ), from Europe Noe MARBEF and

by CNRS UMR 7093. Fieldwork was supported financially

and logistically by the Institut Francais pour la Recherche

et la Technologie Polaires. The authors would like to thank

Dr J. Dolan for his editorial comments and Dr F. Alonzo for

his help during the fieldwork. The work would have been

difficult without the help at sea of the captain and the crew

from the RV La Curieuse. The technical help from P. Le

Jeune, G. Roudaut and N. Coffineau was greatly appreciated.

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