Seasonal changes in the accumulation of polyunsaturated fatty acids in zooplankton

Seasonal changes in the accumulation of polyunsaturated fatty acids in zooplankton

MELANIE HARTWICH"k, DOMINIK MARTIN-CREUZBURG' AND ALEXANDER WACKER'

r DEmR'I'r-.IENT OF 'l'HEORE'\'l(;AI, A(..!.u,\,rlC ECOI.{)C'; INSTITUTE OF BI0CHI~:.\·11STRY AND nIOLOG'~ UNIVERSITY OF POTSDA~I, t\:\1 NEUEN PAI.AIS 10, D-14469 POTSD;\r-.I, CER:VIANY ANU 'lLL'vINOIOGICAL INSTITUTE, UNIVERSITY OF CONS'[i\:'\CE, ~MINI\USTRASSE 252, D-7B464 KONS'!/\NZ, GERMt\NY

*CORRESPONIJING AUTHOR: melanie.hartwich@uni-potsclam.de

Corresponding editor: Marja Koski

In aquatic food webs, consumers, such as daphnids and copepods, differ regarding their accumulation of polyunsaturated fatty acids (PUFAs). We tested if the accumula- tion of PUFAs in a seston size fraction containing different consumers and in DajJ/znia as a separate consumer is subject to seasonal changes in a large deep lake due to changes in the diel<'1lY PUFA supply and specific demands of different consumers.

We found that the accumulation of arachidonic acid (ARA) in Daphnia increased from early summer to late summer and autumn. However, ARA requirements of Daphnia appeared to be constant throughout the yeal~ because the accumulation of ARA increased when the dietary ARA supply decreased. In the size fraction

>

14·0 j-Lm, we found an increased accumulation of docosahexaenoic acid (DBA)

during late summer and autumn. These seasonal changes in DBA accumulation were linked to changes in the proportion of copepods in this size fraction, which may have increasingly accumulated DBA fol" active ovelwintering. 'I'Ve show that con- sumer-specific PUFA demands can result in seasonal changes in PUFA accumulation, which may influence the trophic transfer of PUFAs within the food web.

KEYWORDS: accumulation; DajJhnia; copepods; ARA; DBA

INTRODUCTION

Herbivorous zooplankton provides an important eco- logical link between primary producers and higher trophic levels transferring energy and essential

biochemicals in aquatic ecosystems. This trophic trans- fer varies seasonally with plankton succession and can be affected by the biochemical composition of the food (Gaedke and Straile, 1994; Gladyshev et

a t.,

20 I 1).

Ersch. in: Journal of Plankton Research ; 35 (2013), 1. - S. 121-134 http://dx.doi.org/10.1093/plankt/fbs078

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-215314

Several polyunsaturated fatty acids (PUFAs) have been discussed as essential biochemicals potentially limiting growth and reproduction of herbivorous zoo- plankton in the pelagic food web. PUFAs are struc- tural components of cell membranes and serve as precursors for various bioactive molecules, e.g. eico- sanoids (Stanley, 2000; Heckmann et

a t.,

^2008).

PUFAs are thus important for the fitness of consu- mers, influencing somatic growth, reproduction, immune responses and adaptation to environmental changes (Heckmann et

at.,

2008; Schlotz et

a t.,

2012).

In general, PUFA accumulation (quotient of PUFA concentration between plankton size fractions) was found to increase from small seston particles to zoo- plankton (Kainz et

at. ,

2004; Kainz et

at. ,

2009;

Mariash et

at. ,

20 I I). However, several studies found that the accumulation of PUFAs strongly differs between c1adocerans and copepods. While c1adocer- ans clearly accumulate eicosapentaenoic acid (EPA) from their food, copepods strongly accumulate doco- sahexaenoic acid (DI-IA) (Persson and Vrede, 2006;

Smyntek et

a t.,

2008; Burns et

a t.,

2011; Mariash et

at. ,

2011). Arachidonic acid (ARA) is usually accu- mulated much more in c1adocerans than in copepods (Persson and Vrede, 2006; Smyntek et

at. ,

2008;

Kainz et

at.,

2009). a-linolenic acid (ALA) was found to be accumulated to the same degree in both zoo- plankton groups (Smyntek et

at .,

2008; Kainz et

at.,

2009).

EPA is velY important for freshwater c1adocerans; it affects somatic growth and in particular reproduction (Mliller-Navarra, 1995; Von Elert, 2002; Martin- Creuzburg et

a t.,

20 I 0). Recently, there has been evi- dence that ARA supports Daphnia reproduction similarly to EPA, suggesting that these PUl'l\s are substitutable dietary resources, at least in regard to thei r eflects on reproduction (Martin-Creuzburg et

at. ,

20 I 0; Martin- Creuzburg et

at.,

2012). DHA was found to be the most important PUFA in copepods, increasing egg produc- tion and the hatching success in (marine) copepods (J6nasd6ttir et

ai.,

1995; Arendt et

at. ,

2005; Evjemo et ai., 2008). PUFAs are also needed by zooplankton to adapt to cold temperatures (Schlechtriem et

at.,

2006;

Smyntek et

a t.,

2008; Martin-Creuzburg et

a t.,

2012;

Spelfeld and Wackel; 2012). I-Iowevel;

<

2% of the total fatty acids in c1adocerans were found to be synthesized de 1l0VO (Goulden and Place, 1990), suggesLing that most of the PUFAs detectable in c1adocerans are of dietaly origin.

The availability of dietary PUFAs may change sea- sonally with phytoplankton succession. Different phytoplankton groups are characterized by distin ·t PUFA profiles and! or concentrations of single PUFAs

122

(Ahlgren et

at.,

1990). During spring, phytoplankton is often dominated by EPA-rich diatoms, whereas in summer the proportion of diatoms is usually lower (Hartwich et

at. ,

2012). Seston PUFA concentrations may also be affected by abiotic factors, such as tem- perature or light intensity, which have been shown to be negatively correlated with the PUFA concentration in phytoplankton (Thompson et

at.,

1990; Thompson et

ai.,

1992; Piepho et

at.,

2012). Consequently, the seasonally changing PUFA supply caused by varying taxonomic composition and abiotic factors may influence the fatty acid accumulation of consumers.

The seasonality of PUfA accumulation has not been studied extensively. Kainz et

a t .

(Kainz et

at.,

2008) used four dates in I year to investigate the accumulation of PUFAs in macrozooplankton and reported that the ac- cumulation of PUFAs along a size gradient was highest during spring. Lau et

at.

^(Lau et

at.,

2012) reported t1lat the variation of fatty acid concentrations in nine lakes over three seasons was mainly explained by taxonomic differences and found no seasonal influences. Another study conducted over a growing season, focusing mainly on the correlations between seston and consumer fatty acids, found that the percentage of DBA within total fatly acids in DiaptomuJ showed pronounced seasonality (Ravet et

at. ,

20 I 0). Howevel; until now seasonal changes in PUFA accumulation at different consumer levels have not been studied. Here, we hypothesize that potential seasonal changes in PUFA accumulation may result from (i) adjustments in accumulation to compen- sate for the seasonally varying dietary PUFA supply, or (ii) changes in physiological PUFA requirements of con- sumers, e.g. during different life cycle stages or as an adaption to changing environmental factors such as tem peratu reo

Tn the present study, a high-resolution sampling protocol was used to investigate the seasonality of tile accumulation of PUFAs to evaluate diflerences in PUFA adjustment and demands in different consumer groups.

We used canonical discriminant analyses (CDA) with subsequent multivariate analyses of variance (MANO\'l\) to identify PUFAs discriminating between different seston size (i'actions in oligotrophic Lake Constance. The seasonality of the accumulation of PUFAs was investigated in the seston size fraction

>

140 IJ-m [rom fraction

<

I 00 IJ-m (including

<

30 1J-111) and, as a separate group, the accumulation in Daphnia sp. frol11 seston <30 IJ-I11 (Fig. I). We used a linear 1110del approach to analyse PUl~ accul11ulation in the size fraction

>

14·0 1J-I11, consisting of: various plankton groups, to identify the group(s) l11ainly inAuencing PUFA accul11ulation.

r _{> 140}

flm 1

Copepoda Cladocera

9 aPhniasp,

Ronfers Ciliates

"-

Phytoplankton

)

/' i

< 100 flm r ",'"

<

3O flm

Phytoplankton Phytoplankton

Ronfers Ciliates

Ciliates HNF

Nauplii Bacteria

"- "

^~~

Fig. 1. lliustration of the proposed food web ilHeractions between the difTerelll size fractions > I ~ 0 tLln, < 100 tLln, and < 30 tLln based on the plankton taxa [hal were present in these rC5pective fractions.

DajJ/mia sp. was considered separatcl)~ Arrm·\,s indicate potential fceding interacrions.

METHOD

Study site and sampling

Lake Constance (4·7°40'N, 9°20'E) is a large, deep, warm-monomictic lake (surface area: 4·73 km², <",ca"

=

I 0 I m, <"""

=

254 m) north of the European i\Jps.

Nter strong eutrophication between 1960 and 1980, the lake underwent intensive re-oligotrophication and now approaches its natural oligotrophic state [< 8 JLg phosphorus L - 1 in 2008 during spring circulation

(IGKB, 2008)].

Sampling was carried out weekly from April to November 2008 ·at a long-term sampling site situated in the deep north-western arm of the lake (Uberlinger See, maximum depth = 147 m). "Vater samples were integrated over the depth interval 0- 20 m, which repre- sents approximately the euphotic zone of Lake Constance. Water was filtered through 100 JLm and 30 JLm meshes to obtain the different size fractions <30 and < 100 JLm (i.e. including < 30 JLm). The size frac- tion > 14·0 JLm was sampled with a net haul fi·om 20 to

o

m. From subsamples of plankton > 140 JLm, daphnids were hand-picked for. epa rate analyses.

For determination of particulate organic carbon

(PaC), the different size fractions were filtered onto pre-

combusted glass fibre filters ('vVhatman GF IF, 25-mm diameter) and analysed usil1O" an NCS-2500 analyzer

(ThermoQuest GmbH, Egelsbach, Germany). For the

123

analysis of fatty acids in the different size fractions, subsamples of ~0.5 mg pac were filtered onto pre- combusted glass fibre filters ('vVhatl11an GF IF, 4·7 n""lm diameter). Lipids were extracted, transesterified into fatty acid methyl esters (FAMEs), identified and quanti- fied by gas chromatography (6890N Network GC System, AgiJent Technologies, Boblingen, Germany) according to Wacker and Weithoff (Wacker and Weithoff, 2009) with the following GC configurations:

I JLL of the sample was injected in the split mode (5: I), vaporized in the injector at 250°C and mixed with the carrier gas (helium). FA.tVIEs were separated using a polysiloxane column (Agilent Technologies

J&

W DB-225, 30 m x 0.25 mm x 0.25 JLm) and a tempera- ture gradient (60°C for I min, increasing 20°C min- I until 150°C, 10°C min- I until 220°C, for 13.75 min).

FA.tVfEs were detected using a flame ionization detector at 250°C and quantified by comparison with internal standards and by using multipoint standard calibration curves determined for each FA1VfE from mixtures of known composition. FA1VfEs were identified via known retention times of reference substances · (4 7BB5- U, SupeJco 37 component FA1VfE mix, Sigma-Aldrich, Steinheim, Germany). Double bonds of I B:3* and 18:4·* were identified by comparison with known reten- tion times of different Chlamydomonas extracts containing

I B:3(5,9, 12) and I B:4(5,9, 12, 15) as particular fatty acids (Giroud et at., 198B; Tremolieres, I 99B). The standard calibration curve of 18:3n-6 and 18:3n-3 was used for the quantification of 18:3* and 18:4*, respectively. The detection level was 4·0 ng mg C-I

.

Phytoplankton, ciliates, and rotifers in the different fractions were assessed by the Utermohl technique and crustaceans by light microscopy. For the conversions of organism counts into units of carbon (JLg C L -I), con- version factors determined for Lake Constance or from the literature were used (for details, see Gaedke, 1992).

Statistical analysis

We used CDA with the size fractions < 30, < 100,

> 140 JLm and Daphnia as groups to determine whether PUFAs with 18 and more carbon atoms (calculated as arcsine square root transformed relative proportion) discriminated between the groups. I\. subsequent MANOVA with the potentially important PUFAs identi- fied by the CDI\. was carried out to assess whether dif- ferences in PUFI\. composition (log JLg PUFI\. mg C-I ) between the size fractions and DajJ/znia were significant.

Potential feeding interactions expected between the seston size fractions are illusu·ated in Fig. I. For the size fraction > 140 JLm, we defined the potential food size

as < 100 JLm, because of the various zooplankton

groups ¹¹¹ this fraction, assuming that the whole size range might be covered by their feeding. While Daphnia can take up particles in the range of 1-:10 /Lm (Burns, 1968), copepods can feed on particles in the range of 2-250 /Lm, depending on their developmental stage (Berggreen et at., 1988), and predatOlY rotifers such as As/)lanelllla feed on small rotifers 70 /Lm in length (Sarma and Nandini, 2007). The accumulation in Daphnia was calculated by division with fatty acids (/Lg mg C-^I) in seston <30 /Lm, and the accumulation in the size fraction > 14·0 /Lm was calculated by division with fatty acids in the size fraction < 100 /Lm. We used a one-sample I-test to check if the accumulation of fatty acids was different from I. This gives information on whether a PUFA is concentrated (accumulation factor

> 1), not concentrated (= I) or "diluted" « I) in a con- sumer compared with its food. To analyse seasonal changes' in fatty acid accumulation, the annual cycle was subdivided into difIerent phases. Phase 0 (spring) was excluded from analyses due to the low number of samples. Phase I a (early summel~ n = 7) and Phase 2 nate summer and autumn, n = 9) were used for analyses of differences in PUFA concentration and accu- mulation in the different seston size fractions, because only during these phases were fatty acid data for Dat)/mia available, i.e. no data points were available be- tween 01.07.2008 and 19.08.2008 (mid-summer values, Phase I b).

Differences in fatty acid concentrations (/Lg mg C-^I)

were tested using two-way ANOVA with (i) the size frac- tions < 30, < 100, > 140 /Lm and Daphnia and (ii) the seasonal phase as factors. Differences among levels of factors were evaluated by Tukey's HSD I)osl hoc tests.

Differences in the accumulation of PUFAs between the two "consumer" size fractions (> 140 /Lm, Daj)/mia), and between the two phases were analysed accordingly. To assess which plankton groups in the size fraction

> 140 /Lm were responsible for the observed accumula- tion of fatty acids, we .used a linear model with 'phase' as a factor and arcsine square root transformed propor- tions of the biomass of the different plankton groups as covariate. The separate group 'DaJ)hnia', which per def- inition has a proportion of 100% Daj)/mia, was not included in this calculation. Instead, we used the COI11-

plete data set including the mid-summer values (Phase 1 b), resulting in 11 = 21 for > 140 /Lm.

Since the food quantity may influence PUFA accumulation in consumers, we tested for differences in the quantity (/Lg C L -I) of the fiactions <30 and < 100 /Lm between Phases 1 a and 2 using two-sample I-tests.

A correlation analysis was performed to find potential correlations between the PUFA concentrations in the different size fractions and the concentration in their

respective food (i.e. Daphnia versus seston <::: 30 /Lm,

> 140 versus < 100 /Lm).

Nl analyses were performed using the software package R (version 2.13.0, R Development Core Team, http://www.r-project.org).

RESULTS

Seston biochemical and taxonomic composition

The two-way ANOVA performed to assess differences in PUFA concentrations (/Lg mg C-^I) among the size fractions and between Phases I a and Phase 2 revealed significant differences between the size fractions for all PUFAs considered, but differences between the two phases were significant only for AM and DBA (Table I; Fig. 2). Tukey's HSD posl hoc test revealed a de- crease in the concentration of J\Ri\ in seston

<

:10 /Lm and an increase in the concenU'ation of DBA in the size fraction > 140 /Lm from early summer to late summer/autumn.

PUFA concentrations in DaJ)/mia and in the II'action

> 140 /Lm were not correlated with the respective fatty acids in their food size fraction «30 and < 1 00 /Lm, respectively; SupplementalY data, Table SIlo The results were also not significant when a I-week lag was consid- ered between PUFA concentration in the food fraction and the consumer fraction (Supplementary data, Table SIT).

To determine whether observed increases of PUFA accumulation were based on an increase in food avail- ability, the POC concentration in the food fractions was analysed for differences between the phases investigated. In the fraction

<

30 /Lm, carbon concentrations during early summer (0.26

±

0.05 /Lg C L -I) and late summer/

autumn (0.21

±

0.05 /Lg C L -I) were similar (I = 1.9, df = 7,77, P = 0.09). In the fra tion < 100 /Lm, carbon concentrations also remained at a rather constant level of 0.38

±

0.1 /Lg C L - I in early summer and 0.26

±

0.05 /Lg C L ^{- I} in late summer/autumn (I = 2.6, df=

5,02, P = 0.05; Fig: 3).

In the size Ii-action > 14·0 /Lm, phytoplankton made up 85% of the biomass during Phase I, and in Phase 2 the phytoplankton proportion increased from 6 to 69%

(Fig. 4). The proportion of Daphnia ranged from 2 to 64·% in Phase I and up to 4·5% in Phase 2. Copepods contributed up to 71 % of the biomass in Phase I, whereas in Phase 2 their proportion decreased from 59 to 22%. The proportion of uj)todora showed 1:\-1'0 peaks in the phases investigated (35% in Phase I and 22% in Phase 2, respectively) but was usually < 10%. Rotifers and nauplii contributecl only low proportions of <8%

Table L Results if two-way ANOWl (1) on c oncentrations (log

/-Lg mg

L I) . if diffrrr,ent fatly! cuids usi ng j)/wse (Phase 1 a

=

earlY summer, Pha se 2

=

late summer and autumn) and Slze jracLzons as factors

Results of 2-way AN OVA Size fractions Phase Between fractions

III Within fraction between Phases 1 a and 2

Size fractions x phase Daphnia versus

>140""m Daphnia versus

<30""m

> 140 ""m versus

<100""m

<30 ""m versus

<100""m Daphnia

>140""m

<100""m

<30""m

ALA 0.59 df P-value 3,56 1,56 3,56

>"

>**.

> ¹¹¹¹¹

DGLA

0.31 F P-value F

24.4 7.0

4.6 0.88

1.4 0.93

<"

ARA 0.69 P-value

> 'HHI

>***

>'*

>'

>'

EPA 0.74 F P-value 33.9

5.8 5.6

>11_.

> ~ III

DHA

0.59 F P-value F

51.3 21.1

1.3 1.5

1.1 4.9

<*1111

> 111111

<'

Table section II shows the differences between the fractions and table section III shows the differences between Phases 1a and 2 within each seston fraction, as revealed by Tukey's HSD post hoc test. Greater-than and less-than signs indicate in which fraction or phase a fatty aCid concentration was higher or lower, depending on lhe order of fractions and phases depicted in the third column.

'P< 0.05.

"P<0.01.

"'P<0.001.

in the size fraction > 140 f.1m, and the proportion of ciliates was <0.04-%.

Canonical discriminant analysis

The CDA revealed that the three size fractions and Dail/Illia differed with respect to the different PUFAs. The first canonical axis explained 74% of the variability and was negatively associated with DBA (1'

= -

0.69) and dihomo-'Y-linolenic acid (DGLA; 1'= - 0.67; Table II).

The consumer group Daphnia. was clearly separated fiDm the other groups with respect to DHA and DGIA (Fig: Sa). The second axis explained 2.')% of the variabil- ity and was negatively correlated with EPA (1'= - 0.67).

The third canonical axis explained I % of the variability and was negatively associated with ALA (1'

= -

0.52;

Fig. 5b). The subsequent Mi\NOYA performed with those PUFAs that con-elated best with the canonical axes (ALA, DGIA, ARA, EPA and DHA) confinned that all size fractions and Daphll-ia. were significantly different flDm each other regarding their PUFA concentrations (pillai test statistic: F= 5.2, P< 0.001).

Accumulation

The size fi'action > 140 f.1m and Dajl/l1lia differed regarding their PUFA accumulation. Dail/Illia accumu- lated ALA, ARA and EPA from seston <30 f.1111, whereas DGLA and DBA were not acculllulated

125

(Table III; Fig. 6a, b). In the size fraction > 140 f.11ll, all the PUFAs, except DGLA, acculllulated from

< 100 f.1111 (Table III; Fig. 6c, d).

A two-way ANOYA revealed that the accumulation of PUFAs, with the exception of ALA, differed between the size fraction > 140 f.11ll and Dail/Ill-ia (Table IV), and also suggested significant interactions between the factors 'size fraction' and 'phase' (Phase la and Phase 2) for AM and DHA Tukey's HSD Ilost hoc test showed that the acculllulation of PUFAs, with the exception of ALA, differed between Daphnia and the size fraction

> 140 f.11ll. The accumulation of AM in Dall/mia, as well as the accumulation of DJ-lA in the size fraction

> 14·0 f.1m, was both higher during Phase 2 (late

sumlller/ autullln) than during Phase I a (early summer).

The correlation between PUFA accumulation and the proportion of plankton glDups revealed that the pro- portions of phytoplankton and the observed accumula- tion of PUFAs in the size fraction > 14·0 f.11ll were not correlated, indicating that the acculllulation was not caused by the proportion of phytoplankton (Table Y).

Instead, the PUFA acculllulation was influenced by the zooplankton groups present in this size fraction. The proportion of copepods in this size fraction correlated best with PUFA acculllulation. In general, a higher pro- portion of copepods in the > 140 f.11ll size fraction resulted in a higher accumulation of ARi\ and DJ-lA (Table

. v,

Fig. 7). Moreovel~ a Illore detailed analysis of

0 la lb 2 0 la lb 2 10000

a Total ratty acids b _ALA

Daphnia Daphnia

1000 DGLA EPA

/ ^{RA DHA}

100 /.:

- , ^{... "}

10

.... ~;"T' .... . . . ")..

",

""

,

^....

^,;, , ,

0.1

_{, ,} , , ,

\ 0.01

10000

d

1000 > 140 IJm

100

~ 10

b _I ^I

IJ) I

_

^...

E

, ^{, ,.-}

^...

^"

IJ)

,

_I

2: 0.1 ^{\ I}

c: , I

.Q ~

~ 0.01

~ 1000 e r

8 ~ lJm

< 100 IJm

:2 _u 100

C\l

>.

1

::: C\l

~

^::..'l

~ 10

1

... 1

,,,.'_ -1

_I

_\

_{\ ,;}

/ ... ....

I

...

I 0.1

1000

9..:,JPlJm h< 30 IJ

100

. .

10

J :Y

,

I

... /

_/,

.)- \

- I \ ,;

I .. ,;

0.1 ^I _I

I I 0.01

Apr May Jun Jul Aug Sep Oct Apr May Jun Jul Aug Sep Oct

Fig. 2. Time course of fatty acid concentration (log fJ-g mgC"') in 2008 in Daphnia (a and b), sile fraction > 11·0 fJ-m (c and d), seston

< 100 fJ-m (e and f) and seston <30 fJ-m (g and h). Left-hand panels show n-6 fatty acids, right-hand panels show n-3 fatty acids. Note the different scaling of they-axes. Vertical full lines divide the sampling period into spring ancl clear water phase (0), early and mid-summer (I) and late summer and autumn (2). Phase 0 was not used in any analyses due to a low sample number. Phase I is subdivided into Phase I a and I b depicted with a dasheclline,because during Phase 1b no dara for Dn/)/mia as a separate fraction were available. For values below the detection level, we used zero replacement values (half of the detection level; 20 ng mg C-') for the illustration of fatty acid concentrations.

the data set revealed seasonal differences in the relation- ship between the proportion of copepods and the accu- mulation of ARA, EPA and DHA in the

>

140 fJ..m size fraction. During early summel~ a positive relationship betwee'n copepod proportion and PUFA accumulation

was apparent (Table V, Fig. 7). In contrast, during late summer and autumn, the decreasing proportion of copepods was associated with an increasing accumula- tion of ARl\., EPA and DHA. The proportion of Daphnia in the size fraction

>

14·0 fJ..1ll was related to a

500 0 1. 1b 2

.,

...J <30~m

U < 100 ~m

Cl 400 .=,

c:

""

0

e

₃₀₀

'E 2! _c:

8 c: 200

~ !'l

~ .!!l 100

"

'e 0

a.. C\l

0

Apr MaV Jun Jul Aug Sap Oel Nov

Fig. 3. Seasonal changes of particulate organic carbon concentrations (flg C L -') in the size fractions <30 flm and

< 100 flln in 2008. The vertical lines indicate the seasonal phases as

described in Fig. 2.

higher accumulation of ARJ\ during late summer and autumn (Table V, Fig. 7). The contribution of ciliates to PUFA accumulation in this size fraction could not be calculated, because on 12 dates no ciliates were detected. PUFA accumulation in fraction

>

140 fUTI

was not significantly affected by the proportion of roti- fers. Nauplii proportions correlated significantly posi- tively with i\RA accumulation in general and were positively associated with higher ARi\ accumulation during late summer and spring.

DISCUSSION

\lVe show here that the accumulation of certain PUFAs differs bOtll between tlle size fraction

>

14·0 f.1m and Daj)hnia and between different seasons and that this

Apr May Jun Jul Aug

Table IL Re sults of ^C DA on PUFA proport ions and differ e nt size fractions

Canonical axis 2 3

Explained variability 74% 25% 10/0

Eigenvalue 4.8 1.6 006

R2 0.83 0.62 0.06

P·value <0.001 <0.001 <0.05

Fatty acid Total structure coefficients

C18:3' -0.37 0.33 0.09

C18:3n-6 0.06 0.09 0.32

C18:3n-3 (ALA) 0.25 -0.21 -0.52

C18:4' -0.20 0.49 0.12

C18:4n-3 0.29 -0.18 -0.29

C20:3n-3 -0.28 -0.28 -0.01

C20:3n-6 (DGLA) -0.67 0.20 0.22

C20:4n-6 (ARA) 0.61 0.48 0.23

C20:4n-3 -0.11 -0.11 -0.05

C20:5n-3 (EPA) 0.44 -0.67 0.35

C22:5n-3 -0.63 -0.09 0.36

C22:6n-3 (DHA) -0.69 -0.32 0.20

R2 gives the squarep canonical correlations; total structure coefficients give the correlations between the original variates and the canonical scores. See methods for the identification of 18:3' and 18:4'.

information may be used to draw conclusions on the changing' PUFA requirements of consumers and tl1eir acUustments on PUFA assimilation during tlle growing season. We discuss the hypotlleses: (i) tllat a consumer adjusts tlle accumulation in order to keep tlle body con- centration of a certain PUFA constant in spite of seasonal- ly changing dietaIY PUFA supply, and (ii) tllat a consumer adjusts tlle accumulation because of changing demands.

Daphnia

In Daphnia, the AM concentration per unit carbon was constant throughout the yeal~ whereas the

Sep Oct

Fig. 4. Seasonal changes of the contributions of dinerellt plankton groups in the size fractions > 140 flm to the total plankton biomass conCClllratioll. The lines divide the seasonal phases as described in Fig. 2.

127

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0

3 1.0

a

•

2

20:4n-6

•

^0.5

• _•• •

• _•

0

• _•

^0.0

N

z

_-1

•

~

•

U

...

22:6n-3

•

^-0.5

-2 ^~

\ ...

...

^20:5n-3

•

A

...

-3

...

-1.0

-4 A

-5 -1.5

3 1.0

b

•

_A ^Daphnia

> 140 IJm

...

^A ₀

< 100 IJm

2

...

22:5n-3 ^E1 20:5n-3

• ^x

^{< 30 IJm 0.5}

20:4n-6

.. _• •

^0.0

(")

•

Z 0

~

...

U

••

-0.5

•

-1

x

18:3n-3

•

AX

x •

-2

...

^-1.0

...

-3 -1.5

-4 -2 0 2 4 6 8

CAN1

Fig. 5. CDA plots ror PUE",s (proportion or total ratty acids, arcsine square root transrormed) and dirrerent plankton size rractions. Shown are the first versus the second canonical axis (a) and the first versus the third canonical axis (b). For rurther details see clhble II. See methods ror the derails or the identification or 18:3* and 18:4*

availability of ARJ\ in their food decreased in late summer and autumn. Consequently, the accumulation of ARJ\ in DajJ/mia was higher during late summer and autumn than in early summer. This suggests that the efficiency of AM accumulation increased as a re- sponse to the decreasing dietary i\Ri\ supply and thus supports our hypothesis that DajJ/znia may active- ly adjust their assimilation and/or retention of ARJ\.

A high retention of AM at low dietary supply has already been demonstrated in a laboratory study with Da/Jhnia (Taipale et ai., 20 II). An increased uptake of ARA-rich plankton organisms appears rather unlikely,

128

because daphnids are unselective filter feeders and do not select food particles with regard to their nutritional quality (DeMott, 1986). To some extent, adverse effects associated with seasonal changes in dietary AM concentrations may be counteracted by converting linolenic acid or 22:5n-6 into ARi\ (Brett et

at.,

2006; Taipale et

at.,

2011), a process that may increase with decreasing dietary AM supply. The increased ARA accumulation in Daphnia in late summer and autumn was as well reflected in the size fraction

>

140 J..lm, in which daphnids were also represented.

ALA DGLA ARA EPA DHA

Table III Results if one-samjJle t-test on PUPA accumulation in the difftrent size fractions and in Daphnia

Daphnia >140.,.m

P-value P-value

5.14 <0.001 3.53 <0.01

-0.97 0.35 1.97 0.07

4.94 <0.001 3.66 <001

7.17 <0.001 5.48 <0.001

-0.D2 0.98 3.41 <0.01

For DGLA df = 14, for all other fatty acids df = 15. P-values indicate if accumulation was significant, i.e. diHerent from I. In all significant cases PUFA accumulation was > 1 Ii.e. fatty acid concentration was higher in the consumer fraction than in the food source).

Food quantity is a factor that can influence the trans- fer of energy and fatty acids considerably (Persson et at"

2007; Gladyshev et al., 2011). Thus, changes in food quantity may probably influence the accumulation of PUFAs. When food quantity and PUFA accumulation increase simultaneously while the amount of PUFA per unit carbon in the food remains constant, this could result 'in a higher amount of PUFA per volume avail- able for the consumet~ and does not necessarily indicate

14 0 1a 1b 2

a - -ITotal fat! acids 12 Daphnia

--- i

^OGLA

10 - - lARA

I

I

8 ^I I

I

6 ^I _I

c ^I

.2 4 ^I

:; iii E 2

I

§

₀

CIl 6

"0 C

'0 CIl > 140 ~m

~

5 u.. 4

3 2

Apr May Jun Jul Aug Sep Oct

a changed intensity of accumulation in the consumel: In contrast, a constant or decreased food quantity and a simultaneous increase in PUFA accumulation may indi- cate an active increase of accumulation in the consumer rather than a passive increase by food quantity. The latter was the case in our data, as the carbon concentra- tions of the food fraction

<

30 f.l.m were similar during early summer and late summer/autumn, which sup- ports our conclusion that Daphnia actively increased ac- cumulation of ARA when the supply declined.

In general, the accumulation of EPA in Daphnia was high, which may reflect its physiological importance.

The EPA concentration in DajJ/mia did not correlate with the concentration of EPA in seston <30 f.l.m, The EPA concentration in Daphnia, the EPA concentration in seston, and the EPA accumulation did not differ between early summer and late summer/autumn. The high accumulation and the constant concentration of EPA in DajJ/znia suggest that EPA is subject to homoeo- static regulation. Taking into account that EPA might be converted from AlA or DHA (Weers et at., 1997;

Von Elert, 2002; Martin-Creuzburg et al., 20 I 0), the lack of correlation between EPA concentrations in Daphnia and seston

<

30 f.l.m may result from a high

o

1a 1b 2

14 r.b--=---.-.. ...:. ..

=-.. - ...

IA-:-:LA-:.:;:...-.---='---, 12 Daphnia - - - IEPA 'I

8 6 4 2

8 6

4 2

o

I

I I I I .. I

d

> 140 ~m

- -iOHA :'

I I ,~

I , I I I

I I 1 ' , ^I

:\ ^~ I

^{I I I ,,,}

I I I

'I

^{I!: I J I}

I I, I~ ^I

J':.

^{I.... .. \}

-, I ,'l : : : ", I

;., ,::,':1 ", ,,' .... I,

';;1 ;',tl

~/ ~i

Apr May Aug Sep Oct

Fig. 6. The course of accull1ulation in Da/I/Illin froll1 <30 fJoll1 (a ancl b) ancl accull1ulation in the size fraction > 11·0 fJoll1 froll1 < 100 fJoll1 (c and d). Left-hand panels show n·6 fatty acids, right.hand panels show n-3 fatty acids. Values above the grey line (accull1ulation = I) show positive accumulation, values below this line show dilution. Note the difTerenl scales of they-axes. The vertical lines indicate the seasonal phases as described in Fig. 2.

129

Table IV 'Re sults qf two -wqy ANom ( 1) for the accumulation qf PUFAs and the foctor "phase"

(Phase 1 a

=

e arly summer; Phase 2

=

laLe summe r a nd autumn) and the foc tor "size fi-actions"

(> 140 jJ.m = accumulation in .frac tion > 140 jJ.m .from < 1 00 jJ.m) D aphni a = a ccumula.tion in D aphniafi'O m <30 jJ.m)

ALA DGLA ARA EPA DHA

0.20 0.24 0.55 0.37 0.56

R'

df" P-value F P-value F P-value F P-value F P-value F

Results of two-way Size fractions 1,28 3.9 4.5 17.1 12.7 16.4

ANOVA Phase 1,28 3.2 0.01 13.9 3.7 12.4

Size Iractions x phase 1,28 0.09 4.2 3.4 0.01 6.8

Between fractions Daphnia versus <' ^{> 1111*} >" <It.,.,

>140",m

III Between Phases 1 a Daphnia <"

and 2 >140",m <_.11

Sections II and III show results of Tukey's post hoc tests; section II shows P-values (depicted as stars) for differences in accumulation between the size fractions and section III shows results for differences in accumulation between Phases la and 2 within each size fraction. Greater-than and

less-than signs indicate in which fraction or phase a fatty acid accumulation was higher or lower, depending on the order of fractions and phases in

the third column.

"For DGLA df = 1.27.

'P< 0.05.

"P< 0.01.

'''P< 0.001.

Table V Results qf linear mod els with the accumulation qf PUFAs as de /Jerldent and proportion (prof),;

arcsine square root tmnifrmned) qf differe nt plankton groups in the .fractions > 140 jJ.m as continuous inde pe nde nt variables additionally conside ring ''phase'' as afac tor (Phases 1 and 2)

ALA DGLA ARA EPA DHA

R' P-value F R' P-value F R' P-value F R' P-value R' P-value

df' 0.27 0.23 0.42 0.31 0.54

Cope pod prop. 1,17 0.42 3.8 4.6 2.3 11.3

Phase 1,17 3.5 0.04 0.66 0.48 0.56

Copepod prop. x phase 1,17 2.4 0.93 6.8 4.7 8.1

0.18 0.16 0.32 0.07 0.19

Daphnia prop. 1,17 1.0 1.8 2.9 0,01 0.36

Phase 1,17 2.6 0.8 4.6 1.25 3.0

Daphnia prop. x phase 1,17 0.03 0.4 0.58 a 0.56

0.21 0.02 0.2 0.11 0.17

Phyto prop. 1,17 0.06 0.01 0.05 0.04 0.85

Phase 1,17 3.8 0.16 1.8 1.0 1.5

Phyto prop. x phase 1,17 0.85 0.15 2.5 1.1 1.1

0.21 0.19 0.52 0.2 0.22

Nauplii prop. 1,17 0.06 2.8 6.5 1.6 1.2

Phase 1.17 3.8 1.06 7.2 2.6 3.6

Nauplii prop. x phase 1,17 0.66 0.01 5.0 a ^0.05

0.19 0.17 0.19 0.2 0.2

Leptodora prop. 1,17 1.8 3.1 3.2 3.7 2.8

Phase 1,17 2.2 0.03 0.42 0.15 0.69

Leptodora prop. x phase 1,17 0 0.07 0.49 0.31 0.9

Significant correlatIons between accumulation Clnd proportions were positive. Ciliate proportions in fraction >140 /Jom were excluded from the analysis due to the low number of observations (see Fig. 4). Results for rotifers in > 140 f,<m were not sigOiticant and are not shown. Note that the proportion of Daphnia was derived from the si,e fraction> 140 f,<m (Fig. 4), and accumulation used for correlations were also derived from this size fraction (Fig. 6c and dl.

aFar DGLA df= 1.16.

'P< 0.05.

"P<O.Ol.

130

6 6

"

- - -

"

0 ^{Phase 1} Phase 2 ^{a b}

5 0 5 0

'" '"

j

^{4 0}

^{. g}

^{4 0}

::l ~, ₀ ~ 0

"

E

,

§

³

,

"-

::l 3

, _,

tl

, ,

6~ ^u

"',8

"' "'

~ ^{6 Q)}

,

_~

"

^{0 0}

2

" " ,

2 &-

"

< < ^"-

"

^'0

"

^{tJ:> 0}

^,

" " "

^{0 A}

"

⁰

" " ^" "

^prop.

0 phase prop. x phase

0

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

Daphnia proportion Copepod proportion

6 12

'"

^c

"

^d

5 0 10

I

'"

^I

'"

⁰

0 4 _/0 ⁰ 8 0

~ ~ 0

::l

1 '"

^::l

,

A

E E

,

::l 3 ^{I ::l} 6

,

8 ^{I u 0}

,

}'"

u "-

"' ^"'

^{' , 0 A}

~ ²

'" :f

^{4 0}

,

< "'AJ

'"

^{0 0 A'}

,

dJ.

"

0

'"

^{prop. 2 A}

"

'" _'"

^phase prop. x phase ^~~ A ^{0 0 prop.} prop. x phase

0 0

0.00 0.02 0.04 0.06 0.08 0.10 0.0 0.2 0.4 0.6 0.8 1.0

Nauplii proportion Copepod proportion

Fig. 7. DaIJ/mia, cope pod and nauplii proportions in the size fraction> 140 J.l.m versus AM and DBA accumulation during Phase I (a ancl b) and Phase 2. Dashed and dotted lines indicate changes in accumulation during Phase I and Phase 2, respectively, but are only used to iliustl'ate the results of the ANCOVA (Table V) and do not indicate significant slopes or regression lines. The text in the lower left corners give information on the significant independent variables and interactions, respectively, as depicted in "fable

v.

Note that the data are exclusively derived from the size fraction> 1'10 J.l.m including the proportion of DaIJ/lIIia, which was derived from individuals counted in this fraction (cf.

Fig. 4). Note the different scaling of y-axes.

flexibility in the retention and assimilation of EPA m DajJhnia even to short-term changes in EPA supply.

Size fraction

>

140 fLD1

In the size fraction > 140 ".1,111, the accumulation of DHA increased in late summer/autumn, which was caused by an increase in the DBA concentration in this fraction, while DHA concentrations in its potential food fraction, i.e. size fraction

<

100 f.Lm, remained constant.

The increase in DBA concentrations in the size' fraction

> 140 f.Lm was presumably not caused by the observed higher phytoplankton proportions in autumn. Despite the temporarily higher proportion of phytoplankton in the fraction > 14·0 f.Lm, we found no correlation between phytoplankton and PUFA accumulation

(Table V). Moreovel~ the DBA concentrations that

would have been needed to result in such a high

accumulation as measured in the fraction > 140 f.Lm are usually not found in phytoplankton. In the entire phyto- plankton fraction, a DHA concentration of at least 23 f.Lg mg C-^I would have been required to account for this accumulation, which is much higher than the DHA concentrations of single freshwater algae reported in the literature (e.g. 11 f.Lg mg C-^I in Cryptomonas phaseoLus) (Boechat and Adrian, 2005). The proportion of nauplii in this size fraction was positively associated with AM accumulation. However, compared with other plankton groups, the proportion of nauplii was relatively low

« 5% with one exception), suggesting that this' repre- sents a spurious correlation. The proportion of Daphnia in the fraction> 140 f.Lm was positively associated with higher ARi\ accumulation in late summer/autumn, and the proportion of copepods correlated positively with the increased accumulation of ARi\, EPA and DHf\. This suggests that PUFA accumulation in this