Associating particulate essential fatty acids of the omega3 family with phytoplankton species...

Associating particulate essential fatty acids of the x3family with phytoplankton species composition in aSiberian reservoir

NADEZHDA N. SUSHCHIK,* MICHAIL I . GLADYSHEV,* OLESIA N. MAKHUTOVA,*

GALINA S. KALACHOVA,* ELENA S. KRAVCHUK* AND ELENA A. IVANOVA†

*Institute of Biophysics of Siberian Branch of Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, Russia†Krasnoyarsk State Agricultural University, Krasnoyarsk, Russia

SUMMARY

1. We studied variation in the composition of fatty acids in the seston of a small freshwater

reservoir with changes in phytoplankton composition during four growth seasons. We

focused on the dynamics of the x3 fatty acids because of their potential importance for

zooplankton nutrition.

2. Total diatoms were related to the 20:5x3 fatty acid (eicosapentaenoic, EPA) content in

seston. Among two dominant diatom genera, Cyclotella was not associated with EPA

content. In contrast, there was a significant correlation between Stephanodiscus and the

percentage contribution and content of EPA throughout the study. Hence, freshwater

diatoms can differ strongly in content of the essential EPA.

3. We considered abundant cyanobacteria as a potential source of 18:3x3 fatty acid

(linolenic, ALA) to aquatic food webs. Among four dominant cyanobacteria species, two

(Anabaena flos-aquae and Planktothrix agardhii) showed significant correlation with the ALA

content of the seston, while the other two (Aphanizomenon flos-aquae and Microcystis

aeruginosa) did not.

4. Dinophyta had a relatively high level of 22:6x3 (docosahexaenoic, DHA) for freshwater

species and can be also a source of EPA to aquatic food webs.

5. Our results show that various species of diatoms as well as cyanobacteria can be of

contrasting nutritional value for zooplankton because of their different content of the

essential PUFAs. Diatoms, which are low in EPA, could not be considered as a valuable

food, while some field populations of cyanobacteria might be valuable sources of essential

ALA.

Keywords: cyanobacteria, diatoms, fatty acids, food quality, species composition

Introduction

Fatty acids in particulate matter and sediments are

widely studied as biomarkers of the sources and

transfer of organic matter in aquatic food

webs (Claustre et al., 1989; Ederington, McManus &

Harvey, 1995; Desvilettes et al., 1997; Volkman et al.,

1998). Some of them, namely polyunsaturated fatty

acids of the x3 family (x3 PUFA), are now recognised

as essential for the nutrition of a wide range of

animals (Sargent et al., 1995; Ahlgren et al., 1997;

Gulati & DeMott, 1997; Arts, Ackman & Holub,

2001). The x3 PUFAs in freshwater ecosystems

are produced mainly by micro-algae, with their syn-

thesis depending strongly upon genetic (Cobalas &

Lechado, 1989; Gugger et al., 2002) and environmental

factors, such as temperature, mineral nutrients,

Correspondence: Nadezhda N. Sushchik, Institute of Biophysics

of Siberian Branch of Russian Academy of Sciences,

Akademgorodok, Krasnoyarsk, 660036, Russia.

E-mail: [email protected]

Freshwater Biology (2004) 49, 1206–1219 doi:10.1111/j.1365-2427.2004.01263.x

1206 � 2004 Blackwell Publishing Ltd

light, salinity and others (Pohl & Zurheide, 1982;

Roessler, 1990).

The PUFA content of seston has been considered

to be one of the main factors potentially controlling

the growth of zooplankton in freshwater ecosystems

and, therefore, to be a good predictor of the

nutritional quality of the environment, although its

role in zooplankton nutrition is still uncertain (Gulati

& DeMott, 1997). Among PUFAs, eicosapentaenoic

acid 20:5x3 (EPA) is thought to be central to the

nutrition of herbivorous zooplankton, because corre-

lative evidence suggests that this PUFA limits

Daphnia growth (Muller-Navarra, 1995b; Muller-

Navarra et al., 2000). Diatoms usually contain large

amounts of EPA (Cobalas & Lechado, 1989; Dunstan

et al., 1994; Shin et al., 2000) and, along with the

other EPA-rich algal taxa (cryptophytes and chrys-

ophytes), are thought to be of high quality as food

for herbivores. However, it has been shown that

some marine diatom plankton communities con-

tained little EPA (Morris, 1984; Claustre et al., 1989).

During a seasonal survey of two small reservoirs, we

also found a relatively low percentage of 20:5x3 in

seston, even when diatoms were dominant in early

spring (Sushchik et al., 2003a).

Fatty acid composition within the higher taxa of

algae may be quite variable. For instance, cyanobac-

terial species have been divided into four or five

groups showing different patterns and content of x3

PUFAs (Murata, Wada & Gombos, 1992; Cohen,

Margheri & Tomaselli, 1995). The variation in the

EPA content of diatoms is probably related to

differences between species. Therefore, to estimate

correctly the nutritional value of a diatom community

(as well as that of the whole phytoplankton commu-

nity) its species composition and PUFA content of the

dominant species under natural conditions must be

known. However, most studies of the fatty acids in

micro-algae have been carried out in laboratory

cultures (Ahlgren, Gustafsson & Boberg, 1992; Dun-

stan et al., 1994) or in the seston of marine ecosystems

(Kattner, Gercken & Eberlein, 1983; Morris, 1984;

Mayzaud, Claustre & Augier, 1990; Ederington et al.,

1995; Shin et al., 2000), whereas data on the fatty acids

of the freshwater phytoplankton are sparse (Miyazaki,

1983; Leveille, Amblard & Bourdier, 1997).

Besides the EPA limitation hypothesis, recent

studies emphasise the importance of the whole x3

family of PUFAs in the growth of freshwater

cladocerans (Ahlgren et al., 1997; DeMott & Muller-

Navarra, 1997; De Lange & Arts, 1999). There is also

strong correlative evidence that another x3 PUFA,

a-linolenic acid (ALA, 18:3x3), could be the main

limiting factor for growth of a Daphnia population,

whereas EPA was of moderate significance only

(Wacker & von Elert, 2001; von Elert, 2002). We also

found a strong correlation between the biomass of

Daphnia and the content of 18:3x3 in the seston of a

small reservoir (Sushchik et al., 2003a). According to

the literature, cyanobacteria and green algae in

laboratory cultures may contain large amounts of

this essential PUFA (Ahlgren et al., 1992; Gugger

et al., 2002), although different species may vary

greatly in their content. Therefore, we suggest that

besides the obvious differences in PUFA content

amongst the higher taxa, the nutritional value of the

phytoplankton community with respect to the major

x3 PUFAs may vary significantly because of

different species composition. To check this we

need detailed information on the fatty acids in

freshwater phytoplankton communities, taking into

account species composition and interannual differ-

ences.

We carried out a long-term study of fatty acids in

the seston of a freshwater reservoir, which is charac-

terised by distinct periods of domination by cyano-

bacteria and eukaryotic algae during the growth

season and has a variable species composition. The

aim of the study was to elucidate the relationship

between species composition within the higher taxa

(diatoms and cyanobacteria) and the content of

essential PUFAs of the x3 family in the seston.

Methods

Study site

Bugach reservoir (surface area, 0.32 km2; maximum

depth, 7 m) is near Krasnoyarsk City (Siberia, Russia,

56�05¢N, 92�40¢E) and is used for recreation and

fishing. Detailed descriptions of the reservoir are

given elsewhere (Gladyshev et al., 2001; Trusova &

Gladyshev, 2002; Sushchik et al., 2003a). Hydro-

chemical and hydrobiological measurements were

made weekly from May to September in 1999–2002,

from a site near the middle of the reservoir. Samples

were taken from the surface, at the transparency

depth (near 1 m) and at 3 m with a Ruttner-like 8-L

Fatty acids in a freshwater reservoir 1207

� 2004 Blackwell Publishing Ltd, Freshwater Biology, 49, 1206–1219

sampler and were pooled immediately to form a final

bulk sample.

Field and laboratory measurements

Water temperature and dissolved oxygen were meas-

ured with a meter (N5221; MERA-ELWRO, Warsaw,

Poland, in 1999–2000 and with a MO128; Mettler

Toledo, Schwerzenbach, Switzerland in 2001–02).

Phytoplankton subsamples (0.15–0.5 L) were fil-

tered through membrane filters (Vladipor, Mytischi,

Russia; pore size, 0.75–0.85 lm). Micro-algae were

counted and identified under a light microscope. To

calculate biovolume and wet biomass of algae, they

were equated to appropriate geometrical shapes (or

their combinations) and relevant sizes were measured

using an ocular micrometer.

For diatom identification water samples were cen-

trifuged to concentrate phytoplankton cells. The

material collected was oxidised with a 1% solution

of K2Cr2O7 in concentrated sulphuric acid under

modest heat. For scanning electron microscopy

[(SEM), JSM-25S; JEOL, Tokyo, Japan] samples were

rinsed several times with distilled water and air-

dried. SEM photographs were made under magnifi-

cation ·3500–10 000.

To assay particulate fatty acids, 300–500 mL water

subsamples were first filtered through a 0.08 mm

nylon mesh. The seston was then collected from these

prescreened water samples by vacuum filtration onto

membrane filters (‘Vladipor’ pore size 0.75–0.85 lm),

precovered with a layer of BaSO4 to facilitate the

separation of residues. The filters were dried at 35 �Cfor about 30 min, and the residues then separated

from filters, placed into methanol and frozen.

Further fatty acid analysis is described in detail

elsewhere (Gladyshev et al., 2000). Briefly, lipids were

extracted by chloroform–methanol (2 : 1, v/v) three

times. After evaporation of the solvent, methyl esters

of fatty acids were prepared in a mixture of methanol–

sulphuric acid (20 : 1, v/v) at 90 �C for 2 h.

In 1999–2000, methyl esters of fatty acids (FAME)

were analysed on a gas chromatograph equipped with

a mass spectrometer detector (GCD Plus; Hewlett-

Packard, La Jolla, CA, U.S.A.) with a low polar

column as described earlier (Gladyshev et al., 2000).

In 2001–02, a 30 m long · 0.32 mm internal diameter

capillary column HP-FFAP was used for analyses. The

column temperature programming was as follows:

from 100 to 190 �C at 3 �C min)1, 5 min isothermally,

to 230 �C at 10 �C min)1, and 20 min isothermally.

Other instrumental conditions were as described

elsewhere (Gladyshev et al., 2000) The change of the

column type resulted in a higher resolution and the

detection of more FA isomers, but had no significant

influence on peak abundance. Peaks of FAMEs were

identified by their mass spectra similar to those in the

database (Hewlett-Packard) and to those of available

authentic standards (Sigma, St Louis, MI, U.S.A.). The

position of double bonds in monoenoic acids was

determined by GS-MS of FAME dimethyldisulphide

adducts prepared as described elsewhere (Christie,

1989). To determine double bond positions in poly-

enoic acids, GC-MS of dimethyloxazoline derivatives

of fatty acids was used. The dimethyloxazoline

derivatives of fatty acids were prepared as described

elsewhere (Sushchik et al., 2003b).

Canonical correlation analyses between fatty acids

and phytoplankton was carried out conventionally

(Jeffers, 1981), using STATISTICA software, version

6.0 (StatSoft Inc., Tulsa, OK, U.S.A.). Canonical corre-

lation is a multivariate procedure (a special case of the

GLM) for assessing the relationship between two sets

of variables. The parameters from the tables are

weighted, and the weighted sum scores correlated

against each other.

Results

Phytoplankton biomass and species composition

Seasonal changes in temperature varied substantially

between years, reaching a maximum of about 20–

25 �C in July. The total biomass of phytoplankton

varied in the range of 2–200 mg L)1.

In each year the phytoplankton consisted mainly of

Cyanophyceae, Bacillariophyceae, Chlorophyceae,

Euglenophyceae and Dinophyceae, while the biomass

of Xanthophyceae, Cryptophyceae and Chrysophy-

ceae was much lower (the three latter taxa were

combined and presented as miscellaneous) (Fig. 1). In

general, the seasonal succession of phytoplankton was

similar between years, although interannual varia-

tions in species composition, abundance and the

period of domination of particular taxa were

observed.

Initially the phytoplankton was dominated by

diatoms each spring until early June (Fig. 1). Diatoms

1208 N.N. Sushchik et al.


peaked again in the late August of 2000–02. Two

genera were distinctly dominant in this taxon:

Stephanodiscus and Cyclotella. Each of these genera

was represented by several species, although some of

them were difficult to differentiate under a light

microscope. SEM of acid-treated diatom samples

showed that the main representatives were Stephano-

discus hantzschii Grun., Stephanodiscus minutulus (Kutz)

Cleve and Moller, Cyclotella atomus Hust., and Cyclo-

tella meneghiniana Kutz. (Fig. 2). The cells of S. hantz-

schii and S. minutulus were 11 and 8 lm, respectively,

while those of C. atomus were 6 lm and of

C. meneghiniana somewhat larger, up to 17 lm in

diameter. Because of the identification difficulties

under light microscopy, in further calculations the

combined biomass of species belonging to Stephano-

discus or Cyclotella genera was used. It should be noted

that in our previous paper (Sushchik et al., 2003a)

species of Cyclotella genera were erroneously identi-

fied as the single species, Cyclotella comta (Ehr.) Kutz.

The seasonal dynamics of diatoms belonging to

the two dominant genera, Stephanodiscus and

0

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May May May May JulJul Sep SepJul Se

Date

pJul Sep

1999 2000 2001 2002

Fig. 1 The biomass of major taxa of phytoplankton in Bugach

reservoir. May to October, 1999–2002.

S. hantzschii

S. minutulus

C. atomus

C. meneghiniana

Fig. 2 Scanning electron micrographs of the main diatom spe-

cies of the dominant genera of diatoms: Stephanodiscus and

Cyclotella. Left panels – external view of frustules; right panels –

internal view of frustules. Scale bar – 10 lm for S. hantzschii,

S. minutulus and C. meneghiniana, and – 1 lm for C. atomus.



Cyclotella, differed significantly (Fig. 3). In the first

year (1999) an early spring peak of diatoms was

composed mainly of Cyclotella spp., while in the

other years this was mostly Stephanodiscus spp.

(Fig. 3). Stephanodiscus was moderately abundant in

summer 2000 (Fig. 3), while in the other years it was

very scarce or absent. In contrast, Cyclotella biomass

reached a peak in August in three of the 4 years,

and once in September, with the strongest develop-

ment in mid-summer of 2002 (Fig. 3). The other

species (miscellaneous) accounted for a negligible

fraction of the diatom flora, except for biomass in

August 2001 and in May 2002 (note different scales

of biomass in Fig. 3).

In the summer of each year there was an intensive

‘bloom’ of cyanobacteria (Fig. 1). In 1999–2000 this

started in the middle of June, but in the next 2 years

at the beginning of June. In general, the cyanobac-

terial bloom lasted throughout the summer and into

early autumn, except in the second half of the

summer and autumn of 2002, when a biomanipula-

tion of fish was carried out (Fig. 1). The most

intensive bloom was observed in 2000, when biomass

reached 140 mg L)1. There were four dominant

species of cyanobacteria, which differed in abun-

dance between years: Anabaena flos-aquae (Lyngb.)

Breb., Microcystis aeruginosa Kutz. emend. Elenk,

Aphanizomenon flos-aquae (L.) Ralfs, and Planktothrix

agardhii (Gom.) Anagn.

Anabaena flos-aquae was the main dominant cyano-

bacterium species in each growth season, except for its

relatively low abundance in 2000 (Fig. 4). Aphanizom-

enon flos-aquae developed strongly in 1999 and 2000,

reaching the seasonal maximum after or simulta-

neously with An. flos-aquae. Microcystis aeruginosa was

one of the dominant species among the cyanobacteria

only in 1999 (Fig. 4), being sparse in the following

years. In contrast, P. agardhii was completely absent in

the first year of the study, but abundant at the end of

the summer of 2000, and at the beginning of the

summer of 2001 and 2002 (Fig. 4).

Green algae generally accounted for little of the

total phytoplankton biomass, except in mid- and late-

summer of 2001 (Fig. 1). The green algae were

taxonomically very diverse, but peaks in their biomass

(2000–01) generally consisted of Chlamydomonas sp.

and Pandorina morum (O.Mull.) Bory. The biomass of

euglenoids was usually <5 mg L)1. However, eugle-

noids became numerous in 2001: first, Euglena proxima

Dang. peaked in the middle of June and then

Trachellomonas volvocina Ehr. dominated from late

August until the middle of September (Fig. 1).

0

30

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May Jul Sep May Jul Sep May Jul Sep May Jul Sep

1999 2000 2001 2002

Date

Fig. 3 The biomass of main diatom groups in Bugach reservoir.

May to October, 1999–2002.

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Fig. 4 The biomass of dominant cyanobacteria in Bugach res-

ervoir. May to October, 1999–2002.



Dinophyceae were represented by a single species,

Peridinium sp., throughout the study. It was abundant

in 2001 and dominated strongly in 2002, with a

maximum biomass of 195 mg L)1 in the middle of

September (Fig. 1).

Fatty acids

During the first year (1999) only percentage fatty acid

composition was measured and has been reported

previously (Sushchik et al., 2003a). In the following

3 years both the percentage and absolute concentra-

tions of fatty acids were measured. The total concen-

tration was similar in 2000 and 2002, but somewhat

higher in 2001 (Table 1). The number of different

compounds identified, especially PUFA, was less in

2000, when analyses were carried out with a low polar

column (Table 1). Using a polar column (see Meth-

ods), more than 50 fatty acids were identified in the

seston samples in 2001–02, note that the sums of

contents of some chemically close fatty acids are

presented (Table 1).

Over the entire study, the dominant saturated fatty

acids were 16:0, 18:0, and 14:0. Several odd saturated

fatty acids with straight and branched chains were

found. There were also traces of long-chain (from 20

to 24 carbon atoms) saturated fatty acids. Monoun-

saturated fatty acids (MUFA) were dominated by

16:1x7, 18:1x9 and 18:1x7 (Table 1). Other MUFAs,

such as 16:1x6, 16:x5, 14:1x5, 20:1x9 and others,

appeared only sporadically and accounted for <3.5%

of the total.

Table 1 Fatty acid composition (as a % of the total; total – lg L)1) of the seston in Bugach reservoir (Siberia, Russia). May to October

2000–02

Fatty acids

2000 2001 2002

May June July Aug

September

to October May June July Aug Sep May June July Aug

September

to October

12:0 –* – 0.1 0.2 3.7 0.2 0.2 0.2 0.2 0.4 0.3 0.3 0.8 1.0 0.4

14:0 5.1 4.7 3.9 3.3 10.5 7.4 4.8 3.9 4.6 4.7 9.8 5.1 6.2 9.1 7.0

15:0 2.2 3.9 2.9 2.5 4.3 1.0 1.3 1.0 1.0 1.2 1.6 1.0 1.2 1.5 1.3

16:0 34.6 42.6 36.5 35.4 33.8 25.8 27.2 28.8 26.3 26.9 32.7 29.0 26.0 24.4 20.5

17:0 1.7 2.4 2.4 2.2 1.1 0.8 0.8 0.7 0.8 0.9 0.9 1.1 0.9 0.9 1.0

18:0 14.0 17.8 21.8 18.4 11.4 5.0 6.6 11.5 6.6 6.5 9.7 6.0 5.2 3.1 3.2

20:0 1.5 1.7 4.7 1.9 1.1 1.1 0.6 0.9 0.7 0.8 0.6 0.9 0.6 0.2 0.8

22:0 1.1 0.6 1.4 0.4 0.4 1.1 0.5 0.7 0.6 0.5 0.6 1.0 0.4 0.3 1.0

24:0 0.6 0.4 0.3 0.2 0.2 0.7 0.5 0.4 0.3 0.4 0.6 1.0 0.2 0.4 0.4

R (i+a C15, 17) 1.3 2.3 2.4 2.1 2.6 1.3 2.8 2.8 2.5 3.1 2.0 2.8 3.4 2.5 3.9

R 14:1 – 0.1 0.2 0.1 0.7 0.3 0.3 0.1 0.2 0.2 0.4 0.4 0.2 0.1 0.2

R 16:1 16.2 8.4 5.6 4.9 12.8 21.8 10.7 8.6 8.6 10.5 20.0 9.7 11.1 10.2 8.3

18:1x9 11.8 9.4 9.6 12.9 7.5 3.1 6.8 7.0 6.6 4.9 4.6 4.8 5.1 6.5 5.7

18:1x7 3.8 3.4 4.6 6.0 2.1 1.0 1.9 1.4 1.6 1.9 1.1 1.9 2.4 0.7 2.0

20:1x9 – – – – – 0.2 0.2 0.1 0.3 – – 0.1 – – 0.1

R 16:2 0.8 – 0.1 – 0.2 2.0 1.0 1.9 2.3 2.0 0.5 2.2 1.7 2.2 2.3

18:2x6 1.4 1.3 2.0 4.6 2.2 1.5 4.2 4.5 4.5 3.6 1.5 5.5 3.6 2.0 1.3

R 16:3 – – 0.2 – 0.1 3.2 2.2 1.7 2.4 3.5 1.0 2.8 2.7 5.7 4.7

R 16:4 5.1 1.1 0.9 1.1 1.3 0.8 0.9 1.3 1.4 3.0

18:3x6 1.2 0.2 0.4 0.2 0.2 – 0.1 0.3 0.4 0.3 0.1 0.1 0.3 0.5 0.2

18:3x3 – – – 4.0 3.3 2.6 12.3 10.0 10.1 13.6 3.7 14.3 12.7 3.6 5.1

18:4x3 – – – – – 2.4 3.0 3.7 5.9 3.6 1.6 1.4 2.3 3.0 3.5

R (n.i. 18pu) – – – – – 0.2 0.9 1.5 2.8 2.2 0.1 0.5 1.1 1.0 0.9

20:4x6 0.1 – – – – 0.1 0.4 0.2 0.4 – 0.1 0.5 1.0 1.1 1.0

20:4x3 – – – – – 0.1 0.6 0.3 0.3 0.2 0.3 0.2 0.3 0.3 0.3

20:5x3 2.5 – – 0.1 0.3 9.6 4.1 2.8 4.1 3.1 3.2 2.1 4.9 9.2 11.0

22:5x6 – – – – – 0.1 0.4 0.4 0.4 0.2 0.1 0.6 0.6 0.6 0.9

22:5x3 – – – – – 0.1 0.3 0.1 0.1 – – – 0.1 0.2 0.2

22:6x3 – – – – – 1.4 2.4 2.4 3.1 2.1 1.0 1.7 2.8 7.1 8.0

Total 704.8 329.3 467.2 332.6 1078.5 925.5 755.4 781.6 833.5 377.2 284.2 253.2 158.9 180.2 124.2

*Not detected; R (i+a C15, 17), sum of iso and anteiso C15 and C17 acids; R (n.i. 18pu), sum of not identified C18 PUFA.



The PUFAs comprised acids of the x6, x3, x4 and

x1 families with 16–22 carbon atoms. The seasonal

dynamics of the most important essential x3 PUFAs,

18:3x3, 20:5x3 and 22:6x3, are presented in Fig. 5.

Sixteen carbon PUFAs were very diverse and scarce,

each accounting for 0.5–2%. An increase in 16:3x3

fatty acid occurred in late August and September

2001, corresponding to the strong peak in the biomass

of euglenoids (Table 1; Fig. 1). There was a marked

increase in the percentage of 16:3x4 and 16:4x1 fatty

acids in August to September 2002, when dinophyta

dominated the phytoplankton (Table 1; Fig. 1).

Among 18-carbon PUFAs, 18:3x3, 18:4x3 and 18:2x6

were the most abundant over the entire study. The

essential linolenic acid, 18:3x3, increased in late

August 2000, in mid-summer 2001 and in June to

early July 2002 (Fig. 5). Three more 18-carbon PUFAs

were found occasionally, although their double bond

positions unfortunately could not be identified

(Table 1). In 2000, the only long chain (>18 carbon

atoms) PUFA occurred, EPA, 20:5x3, while in the next

2 years several 20, 22 and 24-carbon PUFA belonging

to the x3 and x6 families were found. In these years,

EPA and docosahexaenoic acid (DHA), 22:6x3, were

dominant among the long chain PUFAs and accoun-

ted for 2–15% and 0.5–15% of total fatty acids,

respectively (Fig. 5).

Canonical correlation analyses of phytoplankton and

essential fatty acids of the x3 family

The seston of Bugach reservoir was enriched in three

main x3 essential fatty acids: 18:3x3, 20:5x3 and

22:6x3 (Fig. 5). Based on the literature, we suggest

that their most probable sources among phytoplank-

ton taxa inhabiting the reservoir are species of

diatoms, cyanobacteria, greens and dinophyta. Evi-

dently, several species may contribute to each of the

PUFAs. To define relationships between phytoplank-

ton taxa and the x3 PUFA content of the seston we

performed canonical correlation analyses of four data

complexes. Two of them used the percentage content

of the PUFAs in the seston (of the total fatty acids) and

the percentage of phytoplankton groups in the total

biomass, while the other two analysed the absolute

concentrations of the PUFAs (lg L)1) and the biomass

of phytoplankton groups (mg L)1).

The first analysis was based on percentages, inclu-

ded data for all 4 years 1999–2002, and had 10

phytoplankton taxa, total diatoms, Stephanodiscus,

Cyclotella, miscellaneous diatom species, total cyano-

bacteria, An. flos-aquae, M. aeruginosa, Aph. flos-aquae,

P. agardhii and total green algae as variables within

the phytoplankton matrix and two x3 fatty acids,

20:5x3 and 18:2x3, as variables within the PUFA

matrix. The results are given in Table 2 and depicted

in Fig. 6. The overall canonical correlation coefficient,

R1, pertaining to the first canonical root, was high

(0.81) and statistically significant (P < 0.001). More-

over, the second canonical correlation coefficient, R2,

was also high and statistically significant (Table 2).

Values of the extracted variance and total redundancy

(59%) also indicated a strong relationship between the

two matrixes, i.e. between the 10 phytoplankton taxa

and the two x3 acids.

0

5

10

15)latot fo %(

AP

Eµ (

AP

EL g

)

0

10

20

ALA

)latot fo %(

µ ( A L

A gL

) A

HD

( %

o f

ot ta

)l

50

100

150

–1

0

50

100

–1

0

5

10

15

May Jul Sep

May Jul Sep

May Jul Sep May Jul Sep

1999

2000

2001 2002

µ

0

20

40

60

AH

Dg

() L–1

Date

Fig. 5 The percentages (of total fatty acids) and content (lg L)1)

in the seston of the main x3 PUFAs: eicosapentaenoic, a-lino-

lenic and docosahexaenoic acids. Bugach reservoir. May to

October, 1999–2002.



Regarding factor loadings (Table 2), only total

cyanobacteria and total diatoms show substantial

loadings (positive and negative, respectively) on the

first canonical factor. Obviously, these variables were

determined largely by the Anabaena and Planktothrix,

on the one hand, and by Stephanodiscus on the other.

For the PUFAs both variables show substantial

loadings. Thereby, the first canonical correlation, R1,

was due to the associations of Anabaena and Plankto-

thrix with 18:3x3 and also to the association of

Stephanodiscus with 20:5x3 (Table 2). The second

significant canonical correlation, R2, was attributable

to a negative association of Aphanizomenon in the

phytoplankton percentage matrix with both x3 FA

species in the PUFA matrix. Considering the phyto-

plankton taxa as the explanatory variable, one could

conclude that only Anabaena, Planktothrix, Stephanodis-

cus and Aphanizomenon contributed significantly to the

canonical correlations of the two sets of variables

compared.

The second canonical correlation analysis was

performed for data from 2001–02, when an additional

x3 PUFA, 22:6x3, was detected. As results of the first

canonical correlation analysis revealed unimportant

variables among phytoplankton groups, which were

not correlated with the canonical variable, these

variables were excluded from the second analysis.

The reduction in the variables also allowed optimising

–3 –2 –1 0 1 2

–4

–2

0

2

PU

FA

% –3 –2 –1 0 1 2

–3

–2

–1

0

1

2

–3 –2 –1 0 1 2Phytoplankton % Phytoplankton %

–3

–2

–1

0

1

2

–2 –1 0 1 2 3

–2

0

2

–4 –2 0 2

–6

–4

–2

0

2

–4 –2 0 2

–3

–2

–1

0

1

–3 –2 –1 0 1 2Phytoplankton conc. Phytoplankton conc.

–4

–2

0

2

PU

FA

con

c.

–3 –2 –1 0 1 2 3

–3

–1

1

3

Matrix No. 1

Matrix No. 2

Matrix No. 3

Matrix No. 4

1st Canonical Axis 2nd Canonical Axis

Fig. 6 Plots of correlations between the two canonical variables

of the first and second canonical roots for four data matrices (see

text for details). Solid lines represent linear approximations.

Note that R2 for matrix No. 3 is insignificant. Matrix No. 1

includes the percentages of 10 phytoplankton groups and of two

x3 PUFAs (of the total) as y- and x-axis variables, respectively;

matrix No. 2 – percentages of five phytoplankton groups and

three x3 PUFAs; matrix No.3 – biomass of nine phytoplankton

groups (mg L)1) and concentrations of two x3 PUFAs (lg L)1);

matrix No. 4 – biomass of five phytoplankton groups (mg L)1)

and concentrations of three x3 PUFAs (lg L)1).

Table 2 Results of canonical correlation analysis of matrix No. 1

of the percentages of two x3 PUFAs (of total fatty acids) in

seston and the percentages of 10 phytoplankton groups (of total

biomass) in Bugach reservoir. Samples were collected in the

period May to October in 1999–2002

Phytoplankton (%) PUFAs (%)

Number of variables 10 2

Variance extracted (%) 26.0 100.0

Total redundancy (%) 16.0 59.0

Number of valid cases 72

Root 1 Root 2

Eigenvalues 0.656 0.533

Canonical R 0.810 0.730

Chi-square 118.0 49.1

d.f. 20 9

P <0.001 <0.001

Phytoplankton (%) Factor loadings

Diatoms )0.393 0.359

Cyclotella )0.108 0.248

Stephanodiscus )0.404 0.275

Miscellaneous diatoms )0.130 )0.069

Cyanobacteria 0.878 )0.024

Anabaena 0.508 )0.314

Aphanizomenon 0.242 0.595

Microcystis 0.066 0.302

Planktothrix 0.447 )0.306

Greens 0.054 0.232

PUFAs (%)

20:5x3 )0.900 )0.437

18:3x3 0.336 )0.942



of the ratio between the number of variables and the

number of cases (observations), because the latter was

reduced to a 2-year set. Furthermore, the dinophyta

Peridinium was added because it could be a source of

22:6x3. Hence, the matrix correlated included

percentages of 18:3x3, 20:5x3 and 22:6x3, for the

PUFAs, and of Anabaena, Planktothrix, Stephanodiscus,

Aphanizomenon and Peridinium, for phytoplankton

percentages. The analysis revealed a very strong

relationship between the two sets of variables

(Table 3; Fig. 6). The overall variance extracted, as

well as total redundancy, was higher than that for the

previous correlation (Tables 2 and 3). Canonical cor-

relations, R1 and R2, pertaining to the first and the

second canonical roots, were both high and statisti-

cally significant (Table 3). In this analysis three

canonical roots were calculated, because there were

three variables in the PUFA matrix. Nevertheless, the

third canonical correlation was small (R3 ¼ 0.162),

statistically insignificant (v2 ¼ 0.92, d.f. ¼ 3) and

thereby excluded from the following interpretation.

Factor loadings (Table 3) indicated that the overall

relationship between the two sets was attributable to

associations between Anabaena and Planktothrix and

18:3x3, as well as Stephanodiscus and Peridinium with

20:5x3 and 22:6x3 in the first canonical root, and an

association between Peridinium rather than Stephano-

discus with 22:6x3 in the second canonical root.

The third correlation analysis included data on

concentrations of 20:5x3 and 18:3x3 fatty acids and

the biomass of phytoplankton groups in 2000–02. The

overall canonical correlation (R1), as well as the

redundancy and variance extracted (Table 4; Fig. 6),

was slightly lower than that of correlation matrix No. 1

(Table 2), but the second canonical root was insignifi-

cant (Table 4). Therefore, an interpretation of factor

loadings of the second canonical root was omitted.

Factor loadings of the first canonical root revealed a

high association of Stephanodiscus with 20:5x3 only, in

contrast to factor loadings of the percentages, where

one more association between Anabaena and Plankto-

thrix and 18:3x3 took place (Table 2).

Correlation No. 4 included data on concentrations

of 20:5x3, 18:3x3 and 22:6x3 in the PUFA matrix and

the biomass of five phytoplankton groups obtained in


of the percentages of three x3 PUFAs (of total fatty acids) in

seston and the percentages of five phytoplankton groups (of

total biomass) in Bugach reservoir. Samples were collected in the

period May to October in 2001–02

Phytoplankton (%) PUFAs (%)


Variance extracted (%) 68.4 100



Root 1 Root 2




d.f. 15 8

P <0.001 <0.001

Phytoplankton (%) Factor loadings


Anabaena 0.632 )0.263


Planktothrix 0.629 0.127

Peridinium )0.728 )0.679

PUFAs (%)

20:5x3 )0.925 )0.189

18:3x3 0.808 )0.241

22:6x3 )0.712 )0.668


of the absolute concentrations (lg L)1) of two x3 PUFAs in

seston and biomass (mg L)1) of nine phytoplankton groups in

Bugach reservoir. Samples were collected in the period May to

October in 2000–02

Phytoplankton

(concentration)

PUFAs

(concentration)


Variance extracted (%) 35.9 100.0



Root 1 Root 2




d.f. 18 8

P <0.001 >0.05

Phytoplankton (concentration) Factor loadings

Diatoms )0.874 )0.069

Cyclotella )0.093 )0.030

Stephanodiscus )0.939 )0.020

Miscellaneous diatoms )0.141 )0.421

Cyanobacteria 0.246 0.120

Anabaena 0.321 )0.584


Microcystis 0.271 )0.655

Planktothrix 0.403 )0.265

PUFAs (concentration)

20:5x3 )0.776 )0.631

18:3x3 0.277 )0.961



2001–02 in the phytoplankton matrix. Total redund-

ancy and the two first canonical R (Table 5; Fig. 6)

were significantly lower than those for the corres-

ponding percentage data of correlation No. 2

(Table 5). The third canonical correlation, R3, pertain-

ing to the third root, was small (0.337) and insignifi-

cant (v2 ¼ 4.1, d.f. ¼ 3, P > 0.05), and therefore

omitted. Factor loadings (Table 5) revealed an associ-

ation between Stephanodiscus and 20:5x3 in the first

and in the second root, and between Planktothrix and

18:3x3 in the first root. A negative correlation between

Peridinium and Stephanodiscus also contributed in the

second root.

In general, the canonical correlations revealed

significantly higher interactions between the percent-

age content of x3 PUFAs and the relative abundance

of phytoplankton, than between their absolute con-

centrations and biomass.

Discussion

The fatty acid composition of diatoms has been

studied widely in laboratory conditions for aquacul-

ture purposes (Dunstan et al., 1994; Brown et al., 1997).

Field studies on fatty acids of marine diatom com-

munities are also numerous (e.g. Claustre et al., 1989;

Mayzaud et al., 1990; Shin et al., 2000), whereas such

data on natural freshwater diatom phytoplankton are

sparse (Leveille et al., 1997; Sushchik et al., 2003a). It is

well known that the species composition of marine

diatoms differs strongly from that of freshwaters, and

the species used in laboratory cultures are limited in

number and rarely the same as natural freshwater

habitants. Moreover, fatty acid composition of the

same species under specific laboratory conditions and

in natural environment may be rather different.

Here we report the seasonal dynamics of particulate

fatty acid composition in a freshwater reservoir,

including several spring periods of strong (almost

100% of total biomass) domination by two wide-

spread diatom genera. The interesting finding is that

the early spring diatom community in the reservoir

could be dominated either by Stephanodiscus or by

Cyclotella spp. and that high concentrations of the

essential EPA in spring seston were recorded only

when Stephanodiscus dominated. Moreover, high and

significant canonical correlations in all four data sets

were associated partly by the strong relationship

between Stephanodiscus and EPA content, whereas

factor loadings for Cyclotella were low for both the

percentage and absolute concentration of EPA

(Tables 2 and 4). Cyclotella species often peaked in

mid- or late-summer, as did Stephanodiscus species on

one occasion, although these summer diatom sub-

populations were probably low in EPA. It is known

that high temperature often leads to a decrease in the

PUFA content of micro-algae (Roessler, 1990; Sushc-

hik et al., 2003c). Higher summer temperature might

also result in a decrease in the PUFA content,

including the EPA, of the diatoms in Bugach. Hence,

the spring population of Stephanodiscus could be a

valuable source of EPA while Cyclotella, which usually

developed in the summer at higher temperature,

seemed to be a poor source of the EPA.

Note that, besides diatoms, there were other poss-

ible sources of EPA in summer, including Eugleno-

phyceae (Sushchik et al., 2003a) and Dinophyceae. In

support of this, the euglenoid Euglena gracilis con-

tained EPA up to 7% of the total FA (Regnault et al.,

1995). The dinophyte alga Peridinium sp. also showed

a relationship with the EPA percentage in the data

from 2 years, but was not correlated with absolute


of the absolute concentrations (lg L)1) of three x3 PUFAs in

seston and biomass (mg L)1) of five phytoplankton groups in

Bugach reservoir. Samples were collected in the period May to

October in 2001–02

Phytoplankton

(concentration)

PUFAs

(concentration)


Variance extracted (%) 67.5 100



Root 1 Root 2




d.f. 15 8

P <0.001 <0.001

Phytoplankton (concentration) Factor loadings


Anabaena 0.374 0.085


Planktothrix 0.567 0.484

Peridinium )0.270 )0.624

PUFAs (concentration)

20:5x3 )0.638 0.528

18:3x3 0.520 0.372

22:6x3 )0.224 )0.035



EPA concentration in the same period (Tables 3 and

5). This could be explained by marked variation in the

fatty acid concentration, as many disparate factors

could occasionally determine the concentration in the

reservoir. Calculation of the percentages of PUFAs

and algae seemed to normalise the data and smoothed

this variation, and hence correlations with the per-

centage composition appeared to be more reliable in

determining the potential sources of the PUFAs.

It has been shown previously that, in laboratory

culture conditions with sufficient P, C. meneghiniana

Kutz. contained only moderate amounts of EPA (6.7%

of total), and even smaller amounts (2.1%) under

P-limiting conditions (Muller-Navarra, 1995a), while

S. hantzschii Grun. was very rich in EPA (24 % of total;

see von Elert, 2002). These species constituted the

diatom community in this study. Thus, our field data

on the essential PUFA content of the representatives

of these two diatom genera were in good agreement

with laboratory data. The EPA percentage values in 17

marine diatoms used in marine aquaculture ranged

from 5 to 30% of total FA (Brown et al., 1997).

Evidently, variation in the essential EPA content

among diatom taxa (including species) is significant.

Therefore, to estimate correctly the nutritional value

of natural diatom plankton, it is necessary to know

those species that are high or low in EPA.

Evidently, EPA is of great physiological importance

for Daphnia, because it is readily accumulated in the

biomass and is converted into other PUFAs (von Elert,

2002). Potentially it can be supplied directly from food

or synthesised from the biochemical precursor, ALA,

which is considered to be strictly essential for most

invertebrates (Tocher, Leaver & Hodson, 1998). How-

ever, the conversion of ALA to EPA, although

widespread among animals, may be too low to meet

the demand for EPA in many species.

In contrast to the EPA-limitation hypothesis

(Muller-Navarra, 1995a,b; Muller-Navarra et al.,

2000), in the recent literature there is accumulating

evidence that another PUFA, a-linolenic acid (Wacker

& von Elert, 2001; von Elert, 2002) or the whole x3

PUFA family, can be the main limiting factor for

zooplankton growth (Ahlgren et al., 1997; DeMott &

Muller-Navarra, 1997; Park et al., 2002). We have

found that the biomass of the two dominant Daphnia

species in Bugach correlated strongly with the content

of ALA in seston in 1999 (Sushchik et al., 2003a).

Recent data have indicated that some Daphnia

populations are able to show relatively high growth

rates without a food source of EPA, provided they are

supplied with ALA as an x3 PUFA (von Elert, 2002).

We suggest that there may be strong differences

between Daphnia species, and also between clones or

populations of a given species, in their requirements

for EPA and C18 x3 PUFAs in the diet. It is likely that

in lakes and reservoirs daphniids with a high demand

for EPA would be abundant when diatoms rich in

EPA are dominant. On the contrary, a phytoplankton

composed of species rich in ALA or other C18-PUFAs

would favour the growth of Daphnia populations with

a high requirement for ALA.

Bugach is a typical eutrophic freshwater reservoir

characterised by strong nuisance blooms in summer

of the ubiquitous freshwater cyanobacteria (Figs 1 &

4). We previously found a relationship between

essential C18-PUFA-ALA and the biomass of cyano-

bacteria and have investigated its distribution among

the dominant species. Evidently, the four main species

of cyanobacteria in Bugach differed in their fatty acid

composition and gave an unequal contribution to the

ALA content in the seston. According to canonical

correlation results, the factor loadings of An. flos-aquae

and P. agardhii were significantly higher than those of

Aph. flos-aquae and M. aeruginosa in both the percen-

tage content and absolute concentration data matrixes

(Tables 2 and 4). Moreover, the significant second root

of correlation No. 1 was explained by the strong

negative association between Aphanizomenon and both

ALA and EPA. Thus, it appeared that An. flos-aquae

and P. agardhii were good sources of essential ALA for

the aquatic food web, whereas Aph. flos-aquae and

M. aeruginosa were not.

There are numerous data in the literature on fatty

acids in the cyanobacteria that form nuisance blooms

in freshwaters, most information originating from

laboratory cultures (Ahlgren et al., 1992). Anabaena

strains were always considered as an ALA-rich group,

in some strains percentage ALA accounted for 41% of

total FA (Vargas et al., 1998; Li & Watanabe, 2001;

Gugger et al., 2002). Planktothrix strains have also been

shown to be high in ALA, up to 30% (Gugger et al.,

2002). Microcystis aeruginosa has been classified previ-

ously to ‘group 4’ according to the Kenyon–Murata

classification of cyanobacteria (Kenyon, Rippka &

Stanier, 1972; Murata et al., 1992), i.e. it can synthesise

the 18:3x3, 18:3x6 and 18:4x3 fatty acids. Reported

concentrations of the C18-PUFAs in this cyanobacte-



rium differ strongly. Some authors have found that

M. aeruginosa contained mainly 18:3x6, rather than

ALA (Ikawa, Haney & Sasner, 1996; Gugger et al.,

2002), while there are contrary data showing high

levels of x3 PUFA compared with x6 PUFA (Ahlgren

et al., 1992; Hayakawa et al., 2002). Aphanizomenon

strains in laboratory cultures were rich in ALA

(Gugger et al., 2002), while Aph. flos-aquae field sam-

ples taken from blooms in lakes contained only

negligible amounts of ALA (Dembitsky, Shkrob &

Go, 2001). Thus, our data on the essential PUFA of

cyanobacteria during blooms in the reservoir agree

with most literature data. We speculate that variations

in the concentrations of the main C18-PUFAs within

the same or taxonomically close species are explained

either by environmental factors or by culture state and

age, and this is particularly true of strains of Micro-

cystis and Aphanizomenon). In any case, potential

variation in the essential PUFA content among strains

and populations should be considered when the

nutritional potential of phytoplankton communities

dominated by the cyanobacteria is estimated.

We suggest tentatively that an appreciable part of

the variation in the ALA concentration might be

explained by the influence of the other ALA-rich taxa,

possibly including the green algae which sometimes

accounted for a relatively large part of the total

phytoplankton biomass of Bugach. However, this

possibility was not supported by canonical correlation

results: the factor loading for the greens was low

compared with those for the cyanobacteria (Table 2).

It has been shown that in invertebrates, including

daphniids, retroconversion of DHA to EPA occurred

(von Elert, 2002). Hence, DHA-rich species of plank-

ton can be a valuable source of essential PUFAs for the

aquatic food web. When Peridinium sp. dominated

strongly in Bugach, the DHA content of the seston

was high, up to 15% of the total fatty acids on some

dates. The freshwater dinophyte Peridinium cinctum

(O.F. Mull.) Ehr. contained up to 11% of DHA in the

total fatty acids (Ahlgren et al., 1992), while most

marine dinophyta usually showed a much higher

content of the PUFA, up to 30–38% (Cobalas &

Lechado, 1989; Parrish et al., 1993). We conclude that

the dinophyte species in Bugach had a relatively high

concentration of DHA for freshwaters, although sig-

nificantly lower than that in most marine species. Our

data also confirmed the presence of appreciable

amounts of EPA in the freshwater dinophyta.

Although having high essential PUFA content, Peridi-

nium is unlikely to be a direct source of PUFAs for

zooplankton, because its size prevents efficient inges-

tion by daphniids. However, its valuable PUFAs

might be transferred to zooplankton indirectly, via

the food chain.

In conclusion, we speculate that freshwater dia-

toms, or some populations of a given species, can

differ strongly in the content of the essential fatty acid,

EPA. Some of the diatoms, which are low in EPA,

could not be considered as a valuable food for

zooplankton. Moreover, there are differences between

populations of cyanobacteria in C18 PUFA content,

and some of them may be rich in ALA. The latter

populations can be an alternative source of the

essential x3 PUFAs to aquatic food webs.

Acknowledgments

We used the GS-MS of the Joint Equipment Unit of the

Krasnoyarsk Scientific the Centre of Siberian Branch

of the Russian Academy of Sciences. The work was

supported by a personal grant (MK-1846.2003.04) for

young candidates of science (PhD) of the President of

the Russian Federation, by award No. KY-002-X1 of

the U.S. Civilian Research and Development Founda-

tion for the Independent States of the Former Soviet

Union (CRDF) and the Ministry of Education of the

Russian Federation, and by grant No. UR-07-01-011

from the Ministry of Education of the Russian Feder-

ation. At the stage of generalisation the work was also

supported by a personal grant from the Russian

Science Support Foundation. We thank Dr S.I. Genkal

for the electron microscopy and identification of major

diatom species.

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(Manuscript accepted 1 July 2004)



Associating particulate essential fatty acids of the omega3 family with phytoplankton species...

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