ORIGINAL ARTICLE

Polar and Neutral Lipid Composition in the Pelagic TunicatePyrosoma atlanticum

Patrick Mayzaud Æ Marc Boutoute ÆRenzo Perissinotto Æ Peter Nichols

Received: 6 February 2007 / Accepted: 17 April 2007 / Published online: 2 June 2007

� AOCS 2007

Abstract Structure and functioning of colonial pyro-

somes are largely undescribed and their lipid characteris-

tics have received limited attention. The aim of this paper

is to fill this gap on one of the dominant species Pyrosoma

atlanticum. Lipid content is tightly coupled to size and

weight. Lipid composition shows a large dominance of

structural polar lipids. Neutral lipids were dominated by

sterols with low levels of acylglycerols and free fatty acids.

Phospholipids show a dominance of PC with intermediate

percentages of PE and DPG. Other constituents (PS, PI,

LPC, sphingolipids) were present at lower levels. Fatty

acid composition of DAG and TAG showed a dominance

of saturated acids (16:0, 14:0), DHA and intermediate

levels of MUFA. Phospholipids were dominated by DHA

with values exceeding 30% of total FA in all categories

except for PI, where lower percentages occurred. Saturated

acids were second in abundance with MUFA showing

intermediate concentrations. Sterols were dominated by 24-

methylcholesta-5,22E-dien-3b-ol with more than 22% of

the total sterol. Cholesterol (cholest-5-en-3b-ol) repre-

sented only 12 % of the total while 24-methylcholesta-

5,24(28)E-dien-3b-ol accounted for 11% of the total ster-

ols. The low levels of triacylglycerols and free fatty acids,

coupled with high concentrations of glycolipids and phy-

toplankton-derived degraded chloropigments, is evidence

of a direct link with the digestive activity and substantiate

the idea of a high physiological turnover as an alternative

to large lipid accumulation. The fatty acid and sterol pro-

files are consistent with a diverse phytoplankton diet, and a

strong contribution of phospholipid classes to energy

needs, including locomotion.

Introduction

Pyrosomas are a small group of holoplanktonic tunicates,

currently including only 8 described species and 3 genera

[1]. They are restricted to the warmer open ocean waters,

between approximately 50�N and 50oS in all oceans [2].

Our knowledge of the lipid composition in tunicates re-

mains fragmentary and detailed reports encompass essen-

tially benthic tunicates, e.g. ascidians [3, 4] as well as

salps, doliolids and appendicularians [5–8]. Knowledge of

the lipid composition of Pyrosoma is limited to total fatty

acid composition of Pyrosoma atlanticum reported by

Culkin and Morris [9] and Jeffs et al. [10]. Both studies

observed that the concentration of total lipids is generally

low and dominated by structural lipids.

Neither of the two studies on P. atlanticum considered

the lipid composition in terms of lipid classes and their

related fatty acid structure. During the last leg of the

ANTARES-4 cruise to the south Indian and the Southern

oceans, substantial numbers of P. atlanticum colonies were

observed in the region north of the Subtropical Front. As

examination of biomarker lipid profiles from different lipid

P. Mayzaud (&) � M. Boutoute

Universite Pierre et Marie Curie,

Observatoire Oceanologique,

UMR-CNRS 7093, LOV, BP. 28,

06230 Villefranche sur mer, France

e-mail: mayzaud@obs-vlfr.fr

R. Perissinotto

School of Biological and Conservation Sciences,

G. Campbell Building, University of KwaZulu-Natal,

Howard College Campus, Durban 4041, South Africa

P. Nichols

CSIRO, Marine and Atmospheric Research,

Castray Esplanade, Hobart, TAS 7001, Australia

123

Lipids (2007) 42:647–657

DOI 10.1007/s11745-007-3066-0

classes may provide more detailed information pertinent to

life cycle adaptation and food web research than is ob-

tained from total fatty acid profiles, the availability of a

sufficient number of samples prompted an investigation of

the lipid constitution with special emphasis on the polar

structural lipids.

Materials and Methods

Sampling

All samples were taken during the ANTARES-4 cruise of

the research vessel Marion Dufresne, during the period 5

January–23 February 1999. The operational area was re-

stricted between approximately 43–46oS and 61–65 oE, in

the Indian sector of the Southern Ocean and the region of

the Agulhas Front (for details see map in Perissinotto et al.,

16). Colonies of P. atlanticum were collected only in the

Agulhas Front region, north of the Subtropical Conver-

gence, during the period 2–16 February at stations G4

(44�21¢03S; 62�33¢37E). Specimens were collected using a

160 cm-diameter Omori Net (1,000 lm mesh, towed ob-

liquely from 200 m to the surface). Specimens were sorted

out immediately, briefly rinsed with distilled water, blotted

dry on sharkskin filter paper and deep frozen in liquid

nitrogen. Samples were kept at –80 �C until return to the

laboratory within 3 months.

Size, Weight Measurements and Lipid Extraction

Entire specimens were placed frozen on crushed ice and

brought to 0 �C. Size (total length = TL) and fresh weight

(WW) were measured prior to lipid extraction according to

the method of Bligh and Dyer [11]. The Bligh and Dyer

method when used as published may not fully extract

neutral lipids. This problem can be overcome using the

same solvent ratios (i.e. one phase), and a longer extraction

time. All lipid extracts were placed under nitrogen at

–70 �C until analysis. Individual dry weight (DW) was

recorded on sub-samples of 6 colonies dried at 70 �C for

six days to obtain the dry weight/wet weight ratio.

Lipid Analyses

Lipid classes were quantified after chromatographic sepa-

ration coupled with FID detection on an Iatroscan

MK V TH 10. Total lipid extracts were applied to SIII

chromarods using a SAS A4100 autospotter set up to deliver

1 ll of chloroform extract on each rod. Analyses were done

in triplicate. 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:diethyl ether:formic acid 97:3:1.5

(v/v). Phospholipids were separated with chloroform:

methanol: NH4 50:50:5 (v/v). Separation of glycolipids was

achieved with chloroform/ethyl acetate/acetone/methanol/

acetic acid/water (60/12/15/16/3/3). Calibration was

achieved using commercial standards (Tripalmitin, 1,2 di-

palmitoyl-rac-glycerol, stearic acid, cholesterol, 3-sn-

phosphatidyl-L-serine from bovine brain, L-a-phosphatidyl

inositol from bovine liver, sphingomyelin from bovine

brain and a-lysophosphatidylcholine from egg yolk were

obtained from Sigma; MGDG and DGDG from spinach

were obtained from Larodan; L-a-Lecithin from egg yolk,

L-phosphatidyl- ethanolamine from egg and cardiolipin

from bovine heart were obtained from Avanti Polar Lipids).

Separation of neutral and polar lipids was achieved on a

preparative scale by column chromatography on silica gel

(Supelcosil A, Supelco). The neutral lipid fraction was

eluted with six column volumes of chloroform, the acetone

mobile compounds were eluted with four volumes of ace-

tone and the phospholipids were eluted with six volumes of

methanol. Each fraction collected was further separated by

thin-layer chromatography (TLC) on pre-coated silica gel

plates (Analtech, Uniplate) and developed with hexane :

diethyl ether : acetic acid, 170:30:2 (v/v) for neutral lipids,

or chloroform : methanol : aqueous ammonia 70:30:4 (v/v)

for glycolipids, and chloroform: methanol: aqueous

ammonia 50: 50: 5 (v/v) for polar lipids. All operations

took place under nitrogen. Lipid classes were visualized

using dichlorofluorescein and identification was achieved

by comparison with standard mixtures. Acetone fraction

was further separated on HPTLC using the same solvent

system as TLC, but with specific detection of glycolipids

made with Bial’s Orcinol reagents (Alltech). No attempt

was made to separate possible plasmalogen phospholipids.

Each band from TLC was scraped off and submitted to

methylation. Fatty acid methyl esters (FAME) of total

lipids were prepared with 7% boron trifluoride in methanol

[12]. FAME were purified on TLC and recovered in hex-

ane. Gas chromatography (GC) of all esters was carried out

on a 30 m length x 0.32 mm internal diameter quartz

capillary column coated with Famewax (Restek) in a Per-

kin-Elmer XL Autolab GC equipped with a flame ioniza-

tion detector (FID). The column was operated isothermally

at 185 �C for FAME. Helium was used as carrier gas at 7

psig. Injector and detector were maintained at 250 �C.

Individual components were identified by comparing

retention time data with those obtained from authentic (Nu-

Check prep) and laboratory standards (capelin : menhaden

oils 50:50). In addition to the examination of FAME as

recovered, a part of all FAME samples was completely

hydrogenated and the products examined qualitatively and

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

major components, 1 to 9% for intermediate components

648 Lipids (2007) 42:647–657

123

and up to ±30% for minor components. Additional GLC

analysis was carried out on a 30 m length · 0.25 mm

internal diameter non polar DB1 column (J&W Scientific)

to resolve the co-elution of 20:1 and 18:5 fatty acids on

Famewax.

Analysis of sterols was conducted on an aliquot of the

separated sterol fraction after conversion to TMSi (trim-

ethylsilyl) ethers using N,O-bis-(trimethylsilyl)-trifluoro-

acetamide (BSFA, 50 ll) at 60 �C for 1 h. GC and GC-MS

analysis of sterols was achieved as described in Jeffs et al.

[10].

Statistical Treatment

Allometric relationships between dry weight (DW) and

length (TL) for the different colonies (DW = a * TLb) were

computed after log–log transformation and model I

regression [13]. Bivariate analyses were made with Stat-

graphics XV. Cluster analysis was based on Bray-Curtis

distance and farthest neighbour clustering (see: 15).

Correspondence analysis [14] was performed on a re-

duced data matrix of fatty acids transformed to relative

frequencies and scaled so that each row (or column) could

be viewed as a row (or column) of conditional probability

distribution. Distances between profiles were computed

with v2 metrics. This distance gives symmetry to the two

sets of data so that each factorial axis associated to the

cluster of variables (fatty acids) corresponds to a factorial

axis of the cluster of observations (phospholipids). Thus, it

was possible to represent simultaneously descriptors and

observations on the plane defined by the factorial axes.

Details on the method and means of interpretation are gi-

ven in Mayzaud et al. [16]. Computation of multivariate

tests was made using the SPAD 5.5 software [17].

Six replicates were used to compute all means and

standard deviations.

Results

Size, Weight and Lipid Relationships

The size of the colonies of pyrosomes collected during the

ANTARES 4 cruise ranged from 13 to 25 cm. The corre-

sponding wet and dry weight varied from 22 to 64 g and

from 1.1 to 3.6 g respectively, with water content

exceeding 90%. Lipid content on a dry weight basis was

generally low and ranged from 5 to 7%.

The weight (DW) relationships with size or lipid content

were established using specimens of different size. The

assumption is that the increase in length and weight should

reflect a fair degree of integration of the number and

metabolism of the different individual zooids constitutive

of the colony. The log-linear regressions between size and

weight (Fig. 1) were highly significant and resulted in an

overall regression equation of:

log DW = –1.804 + 1.69 log TL (R2 = 0.906,

F = 38.36, p = 0.003),

The relationship between DW and total lipids was also

highly significant and is presented in Fig. 1. The resulting

log linear regression is:

log Liptot = 1.898 + 0.839 log DW (R2 = 0.968, F = 120,

p = 0.001).

A relatively low value for the allometric exponent b was

observed, suggesting a slow rate of lipid accumulation with

increasing weight.

Lipid Class Composition

A detailed composition of the lipids from Pyrosoma at-

lanticum is presented in Table 1. Total lipid concentra-

tions (% dry weight) ranged from 4.9 to 6.9 % with a

mean value of 5.7 ± 0.7 %. Neutral lipids were dominated

by sterols with a mean value exceeding 8% of the total

lipids, while low percentages of triacylglycerols (TAG),

diacylglycerols (DAG) and free fatty acids were recorded.

Polar lipids (% total lipids) were dominated by phos-

phatidyl-choline (PC) followed by phosphatidylethanol-

Fig. 1 log–log regressions between dry weight and length and

between lipid content and dry weight for Pyrosoma atlanticum

Lipids (2007) 42:647–657 649

123

amine (PE), diphosphatidylglycerol (DPG) with lower

levels of phosphatidylserine+inositol (PS+PI), sphingo-

myelin and lysophosphatidylcholine (LPC). The lack of

significant relationships between total lipids or lipid class

per unit weight and either size or dry weight may be

related to the small number of data points(n = 6), or to

the lack of direct interactions between body mass and

lipid metabolism.

Two glycolipids fractions were found in concentrations

exceeding 4% which can be attributed to the filter-feeding

mode of the colonies. Their identification on HPTLC using

Bial’s Orcinol reagents, a sugar specific stain, showed that

one fraction displayed the same Rf as commercial MGDG,

while the second one run slightly faster than DGDG with

the solvent system used. The high concentration of ingested

chloropigments (from 0.3 to 1.6 lg colony–1, see Peris-

sinotto et al., 18), suggests that the second fraction also

originated from ingested phytoplankton. As discussed be-

low, the high level of saturation of this later fraction (96%

saturated acids) could explain the difference in migration

as it is more saturated than the higher plant standard. This

would agree with the possibility of a partial post ingestion

modification.

Fatty Acid Composition of the Total and Neutral Lipids

The relative composition of the total fatty acids and the

main neutral lipid classes is presented in Table 2. Fatty

acid composition of the total lipids was driven by the

abundance of polar lipids. Docosahexaenoic acid (DHA,

22:6n-3) dominated largely with more than 36% of the

total fatty acids. Saturated acids were second in abun-

dance (> 30%) with palmitic (16:0) and myristic (14:0) as

major components. Monounsaturated fatty acids (MUFA)

showed intermediate percentages (12%), with 18:1n-9,

16:1n-7 and 18:1n-7 as major contributors. Among PU-

FAs other than DHA, eicosapentaenoic acid (EPA 20:5n-

3) was the dominant pentaene, 18:2n-6, 18:3n-3 and

18:4n-3 dominated their respective group. DAG and TAG

showed a dominance of saturated acids with 16:0 and

14:0, followed by 22:6n-3 with respective percentages of

18.6% and 21.9% of total fatty acids. Both the DAG and

TAG fractions showed relatively high levels of 18:4n-3

(12.5 and 10.5%) and intermediate levels of MUFA with

the same characteristic fatty acids. The two acylglycerols

showed similar fatty acid composition, the only difference

being the larger proportion of myristic acid (14:0) in

DAG. The free fatty acid fraction was considered because

of the high probability that it originates from digestive

hydrolysis. Indeed, DHA showed the highest percentages

with values ranging from 32 to 64% of the total fatty

acids (mean 44.5%) despite the similar concentrations of

free fatty acid contained in each specimen analyzed (3 to

4.5 % of total lipids). Stearic and myristic acids were the

dominant saturated acids with respective ranges of 2 to

15% and 0.6 to 10%. EPA ranged from 3 to 12%, while

oleic acid was the major MUFA with values ranging

between 3.8 and 6.8%. Other MUFA and PUFA included

18:1n-7, 16:1n-7, 18:2n-6, 18:4n-3 and 20:4n-6 with

percentages close to 2%. Odd branched chain acids (iso

and anteiso) exhibited moderately high values in all

neutral fractions, with percentages of iso15:0, anteiso15:0

and iso17:0 exceeding 1%.

Fatty Acid Fraction of the Glycolipids

The fatty acid composition of the two glycolipid fractions

will only be summarized because of the unusual vari-

ability between replicates. The partial composition pre-

sented in Table 3, showed a large dominance of the

saturated acids with 16:0 and 20:0 as main contributors

for MGDG and DGDG respectively. DHA was second in

importance but with values ranging from 2.8 to 20.4% for

MGDG and from 1.1 to 25.9% for DGDG. Hence, the

percent variability in fatty acid composition of MGDG,

based on six replicates, is 13% for the saturated acids,

35% for the monoenes, 82% for 20:5 and 86% for 22:6.

An increasing trend which agrees with the hypothesis of

selective loss of polyunsaturated acids. The second frac-

tion (DGDG-like) showed 40% variability for the satu-

rated acids, 90% for the monoenes, and 120% for most

unsaturated categories. Since the glycolipids originated

from the phytoplankton chloroplast membranes, such ex-

treme variance can only originate from different degree of

digestive degradation.

Table 1 Lipid content and composition in lipid classes of Pyrosomaatlanticum

Pyrosoma atlanticum n = 6

Total lipids (% dry weight) 5.70 ± 0.67

Lipid classes (% total lipids)

Phosphatidylcholine 45.7 ± 2.57

Phosphatidylethanolamine 13.9 ± 1.70

Diphosphatidylglycerol 6.41 ± 1.90

Phophatidylserine + inositol 2.92 ± 1.07

Lysophosphatidyl choline 2.63 ± 0.64

Sphingolipids 2.12 ± 0.17

Glycolipid # 1 MGDG 4.33 ± 1.56

Glycolipid # 2 DGDG like 4.18 ± 1.43

Diacylglycerols 2.28 ± 0.69

Triacylglycerols 3.52 ± 0.80

Free fatty acids 3.92 ± 0.98

Sterols 8.13 ± 0.60

Values expressed as mean ± standard deviation

650 Lipids (2007) 42:647–657

123

Table 2 Fatty acid composition (% total fatty acids) of Pyrosoma atlanticum total lipids and neutral lipid fractions

Fatty acid Total lipids Free fatty acids TAG DAG

Mean % Sd Mean % Sd Mean % Sd Mean % Sd

14:0 6.93 0.27 5.55 4.81 8.72 2.74 15.05 4.47

Iso15:0 0.32 0.02 1.38 1.88 0.79 0.17 1.55 0.28

Aiso15:0 0.45 0.02 1.10 1.48 0.95 0.13 1.22 0.08

15:0 1.40 0.06 1.18 0.87 1.82 0.16 2.12 0.19

Iso16:0 0.30 0.02 0.37 0.36 0.87 0.16 0.61 0.25

Iso17:0 1.01 0.10 1.47 1.85 1.57 0.45 1.22 0.11

Aiso17:0 0.45 0.11 0.61 0.92 0.49 0.18 0.51 0.05

16:0 16.48 0.25 10.03 7.01 16.62 0.48 16.97 0.78

17:0* 0.77 0.07 0.88 1.00 0.85 0.02 0.83 0.23

Iso18:0 0.38 0.03 0.80 1.01 0.71 0.06 0.38 0.10

18:0 1.19 0.04 1.65 2.07 1.55 0.23 1.83 0.58

20:0 0.19 0.02 0.19 0.16 0.14 0.01 0.05 0.04

22:0 0.06 0.02 0.13 0.03 0.01 0.02 0.05 0.02

S saturates 30.40 0.48 25.89 19.34 36.22 2.82 43.17 5.09

16:1n-7 2.51 0.07 1.99 1.42 4.23 0.55 3.56 0.11

16:1n-5 0.29 0.03 0.26 0.21 0.90 0.39 0.87 0.06

17:1 0.49 0.03 0.56 0.23 0.67 0.08 1.15 0.55

18:1n-9 6.78 0.19 5.00 1.57 5.60 0.40 3.44 0.31

18:1n-7 1.70 0.12 2.22 1.51 2.53 0.08 1.47 0.36

18:1n-5 0.09 0.02 0.25 0.30 0.21 0.07 0.20 0.08

20:1n-11 0.18 0.05 0.03 0.02 – – – –

20:1n-9 0.14 0.03 0.14 0.00 0.13 0.01 0.03 0.05

20:1n-7 0.75 0.05 0.45 0.33 0.49 0.02 0.46 0.14

20:1n-5 0.03 0.02 0.12 0.11 0.23 0.04 0.05 0.03

24:1 0.05 0.02 0.06 0.04 0.02 0.01 0.07 0.04

S monoenes 12.45 0.12 11.08 4.04 15.38 1.07 11.30 0.80

18:2n-9 0.05 0.01 0.20 0.27 0.13 0.11 0.17 0.03

18:2n-6 2.34 0.08 1.82 0.49 3.39 0.26 2.72 0.24

20:2n-6 0.13 0.01 0.19 0.11 0.18 0.01 0.16 0.13

S Dienes 2.74 0.06 2.67 1.03 4.67 0.38 3.81 0.33

18:3n-6 0.11 0.01 0.12 0.04 0.20 0.07 0.22 0.03

18:3n-3 1.01 0.02 0.49 0.19 1.76 0.10 2.04 0.21

20:3n-6 0.09 0.01 0.18 0.10 0.09 0.06 0.07 0.02

20:3n-3 0.10 0.03 0.05 0.02 0.08 0.01 0.06 0.01

S Trienes 1.35 0.05 0.91 0.20 2.16 0.05 2.49 0.32

16:4n-3 0.18 0.03 0.18 0.13 0.58 0.02 0.76 0.25

16:4n-1 – – 0.06 0.05 0.13 0.01 0.14 0.02

18:4n-3 3.95 0.14 2.42 0.33 10.53 0.92 12.51 1.02

20:4n-6 2.10 0.01 2.77 1.83 0.82 0.09 0.42 0.05

20:4n-3 0.27 0.01 0.46 0.41 0.34 0.01 0.31 0.06

S Tetraenes 6.56 0.16 6.06 1.59 12.40 0.83 14.14 1.07

18:5n-3 0.29 0.03 0.04 0.03 0.89 0.11 1.15 0.27

20:5n-3 7.18 0.25 7.20 4.67 4.67 0.18 4.17 0.38

21:5n-3 0.33 0.01 0.47 0.25 0.51 0.10 0.55 0.16

22:5n-6 0.19 0.01 0.47 0.26 0.35 0.03 0.04 0.03

22:5n-3 0.75 0.02 0.72 0.61 0.78 0.06 0.52 0.04

S Pentaenes 8.74 0.25 8.91 5.33 7.20 0.25 6.43 0.67

Lipids (2007) 42:647–657 651

123

Fatty Acid Composition of the Total Phospholipids

and Phopholipid Classes

Total phospholipids (PLt), like all phospholipid classes,

were dominated by DHA with percentages exceeding 30%

of the total fatty acids (Table 3), except for PI where lower

percentages were recorded (23.6%). The saturated acids

were second in abundance with palmitic acid and, to a

minor extent myristic acid as the main constituents. PS

showed a slightly different pattern with 16:0, 18:0 and 15:0

as the dominant saturates. MUFA showed intermediate

concentrations with percentages ranging between 12 and

18% in total phospholipids, PC, PE, LPC, DPG and values

higher than 20% in PS and PI (Table 3). Oleic (18:1n-9),

palmitoleic (16:1n-7) and vaccenic (18:1n-7) acids were

the main components, except in PS where 20:1n-7 ac-

counted for 4.2% of the total fatty acids. Pentaenes repre-

sented the fourth largest group in abundance, with EPA as

the main component with values exceeding 15% in PE,

ranging from 7.5 to 9% in PC, DPG and PI. Lower EPA

values were recorded in PC and LPC with percentages of

5–6%. Three other PUFA were present in smaller but

significant concentrations: 18:4n-3 with values above 3 %

in PLt, PC, DPG, LPC and below 1% in the other polar

lipids classes; 20:4n-6 with values above 4% in PE, PI and

below 1.2% in PC, LPC; 18:3n-3 with percentages around

1% in all phospholipid classes.

Contrary to the pattern observed with neutral lipids, odd

and even branch chain acids (iso15:0, iso16:0, iso18:0,

anteiso15:0 and anteiso17:0) exhibited low concentrations

with values below 0.5% of total fatty acid. Iso17:0 showed

higher percentages with values ranging from 0.5 to 1.1,

with maxima in PI and LPC.

Sterol Composition

As indicated earlier, sterols made a large fraction of the

total lipid. A total of 21 sterols were identified in P. at-

lanticum (Table 4). The sterol composition was dominated

by 24-methylcholesta-5,22E-dien-3b-ol with more than

22% of the total sterol. Cholesterol (cholest-5-en-3b-ol),

which usually dominates in most animal tissues, repre-

sented only 12% of the total while 24-methylcholesta-

5,24(28)-dien-3b-ol (24-methylenecholesterol) accounted

for 11% of the total sterols. Additional important constit-

uents ranging from 5 to 9% included: 5a-cholest-22Z-en-

3b-ol (cis-22-dehydrocholestanol), 24-ethylcholest-5-en-

3b-ol (24-ethylcholesterol), cholesta-5,22Z-dien-3b-ol

(cis-22-dehydrocholesterol) and 5a-cholestan-3b-ol (cho-

lestanol). This composition contrasts with the large domi-

nance of cholesterol classically reported for crustaceans

[27] and suggests a large influence of ingested phyto-

plankton as suggested by the high chlorophyll content re-

corded in these specimens Table 5.

Discussion

Pyrosomas are important tunicates that play a major role in

food webs of many oceanic areas [19]. Yet, they are among

the least investigated of all invertebrates, with very little

information on their lipid structure. Their lipid class

Table 3 Partial fatty acid composition limited to the major compo-

nents (% total fatty acids) of Pyrosoma atlanticum glycolipid frac-

tions

Fatty acid MGDG DGDG like

Mean % Sd Mean % Sd

14:0 1.03 0.38 4.39 5.24

16:0 31.54 3.17 10.40 8.34

18:0 8.41 1.74 1.83 1.05

20:0 4.97 1.96 34.45 30.90

22:0 2.68 0.69 10.18 9.12

S saturates 79.20 10.22 69.19 29.032

16:1n-7 1.06 0.21 1.99 1.94

18:1n-9 1.36 0.82 5.08 4.61

S monoenes 6.15 2.15 9.82 8.81

18:2n-6 0.54 0.25 1.46 1.20

S Dienes 1.17 0.24 2.00 2.10

20:3n-3 1.52 0.63 0.04 0.07

S Trienes 2.20 0.28 0.57 0.60

18:4n-3 0.94 0.37 1.19 1.19

S Tetraenes 1.69 0.77 2.21 2.47

20:5n-3 0.97 0.79 3.47 2.51

S Pentaenes 1.34 1.20 4.18 3.12

22:6n-3 8.25 6.96 12.03 12.71

Sd standard deviation, MGDG monogalactosyl diglycerides, DGDGdigalactosyl diglycerides

Table 2 continued

Fatty acid Total lipids Free fatty acids TAG DAG

Mean % Sd Mean % Sd Mean % Sd Mean % Sd

22:6n-3 36.66 0.44 44.47 17.32 21.96 1.06 18.67 2.05

Sd standard deviation, n = 6,–not detected, TAG triacylglycerols, DAG diacylglycerols, * include phytanic acid

652 Lipids (2007) 42:647–657

123

Table 4 Fatty acid composition (% total fatty acids) of Pyrosoma atlanticum total phospholipids (PL) and phopholipid fractions

Fatty acid Total PL PC PE DPG PS PI LPC

Mean Sd Mean Sd Mean Sd Mean Sd Mean Sd Mean Sd Mean Sd

14:0 6.37 0.15 7.69 0.94 1.69 0.67 6.88 0.05 0.87 0.09 0.78 0.35 0.92 0.14

Iso15:0 0.27 0.03 0.31 0.02 0.17 0.02 0.26 0.02 0.19 0.04 0.23 0.07 0.21 0.07

Aiso15:0 0.38 0.06 0.38 0.01 0.63 0.39 0.29 0.01 0.31 0.08 0.90 0.10 0.25 0.06

15:0 1.35 0.04 1.68 0.09 0.59 0.10 1.54 0.03 6.26 1.81 1.27 0.52 0.81 0.10

Iso16:0 0.29 0.06 0.36 0.02 0.16 0.10 0.33 0.02 0.41 0.05 0.35 0.11 0.21 0.05

Iso17:0 0.78 0.11 0.54 0.02 0.69 0.08 0.60 0.02 0.90 0.07 1.10 0.11 1.10 0.08

Aiso17:0 0.37 0.17 0.19 0.01 0.54 0.24 0.17 0.02 0.58 0.09 0.63 0.11 0.43 0.01

16:0 16.21 0.78 15.47 0.19 15.44 0.45 16.85 0.05 11.60 0.07 15.01 0.41 19.19 1.93

17:0* 0.74 0.07 0.59 0.03 0.74 0.04 0.52 0.06 1.53 0.18 1.28 0.08 1.49 0.15

Iso18:0 0.53 0.05 0.39 0.09 0.22 0.03 0.53 0.08 0.78 0.14 0.96 0.22 0.93 0.03

18:0 0.99 0.04 1.06 0.08 0.99 0.20 1.02 0.09 4.73 0.21 4.76 0.03 6.95 0.99

19:0 0.17 0.01 0.14 0.01 0.10 0.03 0.11 0.03 0.76 0.16 1.11 0.36 0.74 0.47

20:0 0.16 0.05 0.12 0.05 0.02 0.01 0.34 0.02 1.19 0.26 0.75 0.04 0.66 0.14

22:0 0.10 0.07 0.02 0.01 0.20 0.01 0.11 0.04 – – 0.19 0.008 0.06 0.01

S saturates 28.77 0.85 29.07 1.10 22.24 1.21 30.60 0.08 30.21 1.56 29.32 1.48 33.96 3.08

16:1n-7 2.65 0.02 2.80 0.10 2.40 0.24 3.84 0.10 0.67 0.02 2.09 0.03 1.71 0.06

16:1n-5 0.36 0.09 0.48 0.07 0.27 0.10 0.55 0.03 0.28 0.12 0.35 0.06 0.39 0.03

17:1 0.48 0.02 0.52 0.02 0.74 0.07 0.63 0.03 0.80 0.05 1.49 0.11 0.72 0.06

18:1n-9 6.49 0.21 5.03 0.17 10.13 0.41 8.92 0.97 10.50 0.98 13.29 0.24 7.59 0.90

18:1n-7 1.96 0.12 2.11 0.25 2.45 0.48 1.87 0.65 3.54 0.34 4.05 0.26 2.48 0.08

18:1n-5 0.08 0.04 0.11 0.04 0.09 0.06 0.17 0.04 0.54 0.13 0.46 0.06 – –

20:1n-11 0.19 0.03 0.18 0.05 – – 0.19 0.12 – – – – – –

20:1n-9 0.34 0.11 0.13 0.01 0.10 0.02 0.23 0.11 1.20 0.06 – – – –

20:1n-7 0.80 0.03 0.97 0.08 0.15 0.04 1.62 0.16 4.22 1.25 1.62 0.45 2.49 0.34

S monoenes 13.64 0.20 12.45 0.12 16.54 0.18 18.18 0.65 23.64 1.11 24.91 1.96 15.46 1.03

18:2n-6 2.28 0.11 2.34 0.14 1.70 0.09 2.61 0.03 1.81 0.15 2.54 0.22 2.08 0.05

20:2n-6 0.24 0.05 0.16 0.03 0.04 0.02 0.14 0.10 0.23 0.02 0.50 0.23 – –

S Dienes 2.56 0.11 2.57 0.13 1.79 0.04 2.81 0.09 2.04 0.13 3.05 0.36 2.08 0.05

18:3n-6 0.11 0.01 0.20 0.04 0.02 0.01 0.20 0.01 – – – – – –

18:3n-3 0.98 0.06 1.17 0.10 0.60 0.08 0.88 0.05 0.62 0.06 0.70 0.069 0.80 0.13

20:3n-9 0.07 0.03 0.05 0.00 0.03 0.01 – – – – – – – –

20:3n-6 0.11 0.02 0.12 0.01 0.05 0.03 0.29 0.09 0.04 0.01 0.54 0.41 – –

20:3n-3 0.07 0.00 0.07 0.03 0.02 0.02 0.01 0.01 0.05 0.04 – – – –

S Trienes 1.35 0.11 1.60 0.10 0.73 0.06 1.39 0.06 0.71 0.07 1.27 0.44 0.80 0.13

18:4n-3 3.86 0.33 5.43 0.36 0.95 0.09 3.30 0.05 1.70 0.39 1.75 0.08 3.73 0.19

20:4n-6 2.16 0.07 1.19 0.08 5.92 0.29 2.85 0.38 2.10 0.04 4.54 0.37 0.78 0.16

20:4n-3 0.27 0.01 0.36 0.01 0.13 0.02 0.25 0.00 0.25 0.02 0.24 0.11 0.33 0.01

S Tetraenes 6.36 0.28 7.36 0.41 7.14 0.30 6.44 0.35 4.31 0.53 6.52 0.41 4.84 0.36

18:5n-3 0.36 0.04 0.31 0.09 0.11 0.02 – – – – – – – –

20:5n-3 7.44 0.26 5.74 0.26 15.91 0.48 8.38 0.06 4.68 0.32 10.10 0.37 4.33 0.74

21:5n-3 0.32 0.01 0.40 0.02 0.10 0.02 0.25 0.02 0.51 0.18 0.11 0.03 0.46 0.01

22:5n-6 0.18 0.04 0.15 0.02 0.21 0.02 0.11 0.04 0.52 0.16 0.77 0.38 – –

22:5n-3 0.76 0.04 0.73 0.06 0.43 0.09 0.55 0.03 0.60 0.04 0.33 0.05 0.61 0.19

S Pentaenes 9.07 0.18 7.33 0.14 16.77 0.52 9.29 0.04 6.32 0.38 11.31 0.10 5.40 0.92

22:6n-3 38.24 0.33 39.62 0.67 34.80 0.70 31.29 0.35 32.77 0.94 23.62 2.45 37.46 2.66

Sd standard deviation, n = 3, – not detected, PC phosphatidylcholine, PE phosphatidylethanolamine, DPG diphosphatidylglycerol, PSphosphatidylserine, PI phosphatidylinositol, LPC lysophosphatidylcholine, * include phytanic acid

Lipids (2007) 42:647–657 653

123

composition is unknown, but the dominance of phospho-

lipids is in agreement with data on other subtropical pelagic

tunicates [6,7,10]. The low levels of triacylglycerols and

free fatty acids, coupled with high concentrations of

glycolipids, is also in agreement with the observation made

by Deibel et al. [6] on a different pelagic tunicate, the

larvacean Oikopleura vanhoeffeni. The concentration of

glycolipids is likely related to the high levels of chloro-

pigments (present as degradation products) recorded in

Pyrosoma atlanticum [18] and substantiates the idea of a

high physiological turnover as an alternative to large lipid

accumulation. Indeed, pyrosomas are known to graze on a

wide range of microplanktonic taxa, ranging from sub-

micronic particles to large diatoms, and in warm oligo-

trophic waters are facing relatively continuous food supply.

The presence of a pool of free fatty acids coupled with two

phytoplankton glycolipid fractions seems indicative of

such feeding activity and digestive processes [18]. The

presence of phospholipids in excess of 70% of the total

lipids seems a general feature of gelatinous pelagic

organisms and indicative of investment in cell membranes

as reported for ctenophores [20, 21], larvaceans [6] and

chaetognaths [22, 23]. In terms of polar lipid constituents,

P. atlanticum follows the pattern described for most marine

zooplankters, with PC as the main component followed by

PE and DPG [24].

A comparison of the total fatty acid pattern among

samples collected at different locations in the subtropical

Atlantic Ocean by Culkin and Morris [9], the Pacific Ocean

by Jeffs et al. [10] and the Indian Ocean in the present

study shows a relatively similar composition, with very

high levels of DHA, palmitic, oleic and myritic acids and

EPA as main descriptors. Lower percentages of myristic,

palmitic and palmitoleic acids associated with higher levels

of DHA were observed in the present study, compared to

the data of Culkin and Morris [9]. The variation in the fatty

acid profiles may be attributed to differences in the diet of

animals, temperature or to the analytical procedures. A

cluster analysis of all data on P. atlanticum and other pe-

lagic tunicates, such as Salpa cylindrica and Dolioletta

gegenbauri, separated the older data from the more recent

ones (Fig. 2), irrespective of the taxonomic group consid-

ered. Interestingly, the pelagic tunicate Dolioletta ge-

genbauri was clearly separated from P. atlanticum for both

recent studies, based on higher levels of myristic, palmi-

toleic and arachidic acids and lower percentages of pal-

mitic and oleic acids. The differences between the two taxa

are likely related to the differences in phospholipid levels

and composition. Higher percentages of polar lipids in

Pyrosoma is translated in the total lipid fraction into lower

myristic, palmitoleic and arichidic acids, which accounts

for lower percentages than in neutral lipids.

The analysis of lipids in marine animals can often pro-

vide valuable insights into the trophic interactions between

primary consumers and their food supply (see review by

Dalsgaard et al., 25). Because tunicates are known to retain

particles from less than 1 lm in diameter to phytoplankton

cells larger than 10 lm size range [26], the presence of

fatty acid markers of bacteria and phytoplankton in the

neutral fractions (TAG, FFA and DAG) is consistent with a

very diverse diet. In our study, diatom markers (16:1n-7;

16:4 PUFA, 20:5n-3) were recorded but in small propor-

tion, illustrating either a small contribution to the total

ingestion or a very high turnover rate. Prymnesiophytes

and dinoflagellates markers (C18 PUFA including 18:5n-3,

22:6n-3) dominated suggesting a large contribution of

these two groups to the diet of P. atlanticum. Contribution

of bacterial markers (iso and anteiso acids, 18:1n-7) was

also present with relatively high percentages of iso 17:0

and 18:1n-7. An alternate approach was developed in

Perissinotto et al. [18], using multivariate discriminant

analysis, which coupled all data published on phyto-

plankton fatty acids and the pattern of both TAG and FFA

Table 5 Sterol composition of Pyrosoma atlanticum as % of total

sterols

Sterol %

24-norcholesta-5,22E-dien-3b-ol 1.9

24-nor-5a-cholest-22E-en-3b-ol 0.2

cholesta-5,22Z-dien-3b-ol 6.7

27-nor-24-methylcholesta-5,22E-dien-3b-ol

5a-cholest-22Z-en-3b-ol 0.4

cholesta-5,22E-dien-3b-ol 8.3

5a-cholesta-22E-en-3b-ol 2.2

cholest-5-en-3b-ol 11.7

5a-cholestan-3b-ol 5.6

24-methylcholesta-5,22E-dien-3b-ol 22.7

24-methyl-5a-cholest-22E-en-3b-ol

5a-cholest-7-en-3b-ol 3.1

24-methylcholesta-5,24(28)-dien-3b-ol 11.1

24-methyl-5a-cholest-24(28)-en-3b-ol 3.2

24-methylcholest-5en-3b-ol

24-methyl-5a-cholestan-3b-ol 0.4

23,24-dimethylcholesta-5,22E-dien-3b-ol 1.4

24-ethylcholesta-5,22E-dien-3b-ol 3.9

24-ethyl-5a-cholest-22E-en-3b-ol 0.8

24-ethylcholest-5-en-3b-ol 8.3

24-ethylcholesta-5,24(28)E-dien-3b-ol 0.7

24-ethyl-5a-cholestan-3b-ol 1.2

24-ethyl-5a-cholest-24(28)Z-en-3b-ol 0.6

Others 5.4

Total 99.9

* C24 stereochemistry not determined in this study, hence only sys-

tematic names rather than trivial names are given

654 Lipids (2007) 42:647–657

123

acids recorded for the present samples. Results confirmed

that both dinoflagellates and prymnesiophytes are domi-

nant constituents in the ingested material. One question

always open with colonial organisms is whether or not

ingested chlorophyll has passed the plasma membrane of

each individual. The actual process of ingestion and

digestion is a complicated one and no direct microscopic

observation could be made during this study. However, as

shown by Perissinotto et al. [18], most chloropigments

were in the form of pheaopigments (80 to 90%) with a high

rate of degradation into non-fluorescent compounds. Such a

process only occurs during gut transit and strongly support

the hypothesis that the phytoplankton cells were taken up

by individual zooids and digested.

Information on sterol composition is scarce and the

present data can only be compared with those of Jeffs et al.

[10] for samples of Pyrosoma atlanticum and Makar’eva

et al. [27] for samples of Pyrosoma giganteum. The pat-

terns of sterols recorded for P. atlanticum show both

similarities and differences. Both profiles show dominance

of 24-methylcholesta-5,22E-dien-3(-ol, cholesterol, cis-22-

dehydrocholesterol and similar levels of 24-ethylcholesta-

5,22E-dien-3(-ol, 24-ethylcholest-5-ene-3(-ol and 24-

nordehydrocholesterol. However, our specimens from the

Indian Ocean exhibit higher percentages of 24-methylene

cholesterol and cis-22-dehydrocholestanol. They also lack

in some of the major sterols observed off the New Zealand

coast, e.g. 24-methylcholesterol and dinosterol. The dif-

ferences are likely related to the heterogeneity of particles

ingested at the two sites, which may differ substantially in

their phytoplankton and microheterotroph-derived sterols.

We have no knowledge of the sterol metabolism of tuni-

cates, but the low level of cholesterol observed in P. at-

lanticum may suggest that the ability to convert

phytosterols to cholesterol, reported for many crustaceans

[28] may not be as effective in P. atlanticum. Copepods,

euphausiids, ostracods, phyllosomes and other inverte-

brates, such as siphonophores and ctenophores, show a

large dominance of cholesterol in contrast to tunicates

(ascidians and pyrosomes), which contain mostly algal-

derived sterols (10, present study). In terms of trophic

linkage sterol markers of diatoms (22-dehydrocholesterol,

cholesterol, 24-methylenecholesterol), prymnesiophytes

and dinoflagellates (24-methylcholesta-5,22E-dien-3(-ol,

24-ethylcholesta-5,22E-dien-3(-ol, 24-ethylcholest-5-ene-

3(-ol, 24-methylenecholesterol) are present but as indicated

by Volkman [29], many sterols are widely distributed and

assignment to a specific algal class remains difficult. The

lack of dinosterol in our samples may seem contradictory

with the trophic link described earlier, but a number of

dinoflagellate species do not contain dinosterol [30,31].

Hence, it may be that species of dinoflagellates lacking

dinosterol dominated this component of its diet. In oligo-

trophic waters, such as those studied in this paper, the

sterol composition would tend to support a diet dominated

by dinoflagellates and prymnesiopytes [29].

Our knowledge of the phospholipid composition of pe-

lagic marine organisms is, to a large extent, limited to a

description of the main classes in crustaceans: i.e., cope-

pods, euphausiids, decapods [24– 34], and cnidaria (see

review by Joseph, 35). Reports of fatty acid composition is

generally limited to benthic ascidians and total polar lipid

fraction [4], thus preventing meaningful taxonomic com-

parisons. Differences between fatty acid composition of the

phospholipid constituents seem directly related to the

specificity in the pathways of synthesis involved. In this

study, a comparison of the various profiles was achieved

using a factorial correspondence analysis (FCA). The first

three factorial axes accounted for more than 90% of the

total variance and projection of both fatty acid descriptors

Fig. 2 Cluster analysis of all published total fatty acid profiles for

pelagic tunicates based on computed Bray-Curtis distance (x axis non

dimensional). Symbols: P. atlant #= Pyrosoma atlanticum from the

present study; P.a_Jeffs # = Pyrosoma atlanticum from Jeffs et al.

(2004); P.a._Culkin = Pyrosoma atlanticum from Culkin and Morris

(1970); Dolioletta = Dolioletta gengenbauri from Pond and Sargent

(1998); S. cylindrica = Salpa cylindrica from Culkin and Morris

(1970)

Lipids (2007) 42:647–657 655

123

and phospholipid constituents, on the plane defined by the

first two axes (Fig. 3), discriminated between two groups.

The first axis separated PC, PE, DPG on the one hand and

PS, PI on the other, whereas it opposed PC to PE and PS to

PI on the second axis. This is consistent with the concept of

main interrelated synthetic routes via CDP-choline and

CDP-ethanolamine leading to PC and PE and secondary

transformations from PS to produce PE, and from CDP-

diacylglycerol to produce PI [36]. In terms of fatty acid

profiles, differences in PC were linked with 18:4n-3, 18:3n-

3, 20:3n-3, 18:3n-6 and 14:0, while differences in PE were

associated with EPA, arachidonic acid and 22:0. Discrim-

ination between PI and PS were related respectively to

arachidonic acid, 17:1, oleic and cis vaccenic acids and to

20:1n-7, 21:5n-3, 20:1n-9, 20:0 and 15:0.

The fatty acid composition of all polar lipid classes of P.

atlanticum also shared several common features: a high

degree of unsaturation with more than 35% of EPA and

DHA; a relatively constant percentage of DHA; and similar

levels of saturated acids, although with different constitu-

ents. Phospholipids containing high levels of DHA are

often associated with the need for high energy processes.

They have been proposed to be required as conformational

co-factors for the functional assemblage of membrane

proteins, ion pumps and/or complexes of the mitochondrial

electron transport chain [37, 38]. In the specific case of P.

atlanticum, locomotion is slow as a consequence of a slow

continuous jet emerging from the cloacal aperture of the

colony. The jet is produced by the ciliary activity of each

individual zooid, which is under nervous control [2]. We

have no knowledge of the energy needed and the complex

ion and electron transport processes involved in this

motion, but data agree with the hypothesis of a strong

contribution of all phospholipid classes. Within the

membrane bilayer system, PC, PE and DPG are often

associated with both passive and active transport, while PS

and PI are linked to nervous stimulation and PI hydrolysis

is associated to the production of intracellular messengers.

Such a view is consistent with the processes controlling

locomotion and the regulation of buoyancy, which utilizes

sulphate exclusion [2].

In summary, new results for the lipid class and com-

ponent fatty acid and sterol distributions of the gelatinous

pelagic tunicate P. atlanticum collected from the Indian

Ocean sector of the Southern Ocean have been obtained.

The profiles are consistent with a high physiological turn-

over, diverse phytoplankton diet, and strong contribution of

phospholipid classes to energy needs, including locomo-

tion.

Acknowledgements We thank the French CNRS, the French Polar

Institute (IPEV), the South African NRF and the University of

KwaZulu-Natal (Durban, South Africa) for providing funds for this

study. Finally, we like to thank the captain, officers and crew of the

‘‘RV Marion-Dufresne’’ for their assistance and cooperation during

the ANTARES-4 voyage.

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