Species interactions and energy

transfer in aquatic food webs

Jens Munk Nielsen

©Jens Munk Nielsen, Stockholm University 2015

Cover photos: © Jens Munk Nielsen, except bottomright:

©Helena Höglander

ISBN 978-91-7649-316-8

Printed in Sweden by Holmbergs, Malmö 2015

Distributor: Department of Ecology, Environment and Plant

Sciences

ABSTRACT

Food webs are structured by intricate nodes of species interactions which

govern the flow of organic matter in natural systems. Despite being long

recognized as a key component in ecology, estimation of food web

functioning is still challenging due to the difficulty in accurately measuring

species interactions within a food web. Novel tracing methods that estimate

species diet uptake and trophic position are therefore needed for assessing

food web dynamics.

The focus of this thesis is the use of compound specific nitrogen and carbon

stable isotopes and molecular techniques for assessing predator-prey

interactions and energy flow in natural aquatic ecosystems, with a particular

focus on the species links between phytoplankton and zooplankton.

The use of δ15

N amino acid values to predict organism trophic position are

evaluated through a meta-analysis of available literature which included

measurements from 359 marine species (article I). Through a controlled

feeding study isotope incorporation in aquatic organisms, across both plant-

animal and animal-animal species linkages is further assessed (article II).

These studies showed that δ15

N amino acid values are useful tools for

categorizing animal trophic position. Organism feeding ecology influenced

nitrogen trophic discrimination (difference in isotope ratio between

consumer and diet), with higher discrimination in herbivores compared to

omnivores and carnivores (article I). Nitrogen isotope trophic discrimination

also varied among feeding treatments in the laboratory study (article II). The

combined findings from articles I & II suggest that researchers should

consider using group specific nitrogen trophic discrimination values to

improve accuracy in species trophic position predictions.

Another key finding in the controlled laboratory study (article II) was

consistently low C isotope discrimination in essential amino acids across all

species linkages, confirming that these compounds are reliable dietary

tracers.

The δ13

C ratios of essential amino acids were applied to study seasonal

dynamics in zooplankton resource use in the Baltic Sea (article III). Data

from this study indicated that zooplankton assimilate variable resources

throughout the growing season. Molecular diet analysis (article IV) showed

that marine copepod and cladoceran species ingested both autotrophic and

heterotrophic resources.

Evidence from both articles III & IV also revealed that zooplankton feed on

a relatively broad range of diet items but not opportunistically on all

available food sources. Mesozooplankton feeding patterns suggested that

energy and nutritional flows were channelled through an omnivorous

zooplankton food web including microzooplankton prey items. Overall the

results of this thesis highlight that stable isotope ratios in specific

compounds and molecular techniques are useful tracing approaches that

improve our understanding of food web functioning.

LIST OF ARTICLES

Article I Nielsen J.M, Popp B, Winder M (2015) Meta-analysis of

amino acid stable nitrogen isotope ratios for estimating

trophic position in marine organisms. Oecologia Vol. 178:

631-642

Article II Nielsen J.M, Reutervik K, Winder M, Hansson S (in prep)

Amino acid stable isotope discrimination in diverse aquatic

food chains.

Article III Nielsen J.M, Winder M (2015) Seasonal dynamics of

zooplankton resource use revealed by carbon amino acid

stable isotope values. Marine Ecology Progress Series Vol.

531: 143-154

Article IV Nielsen J.M, Fusilli M, Esparza-Salas R, Dalén L, Winder M

(in prep) Marine zooplankton diet preferences across species,

life stages and seasons using DNA barcoding.

Author contributions: Main contributions to study design, field sampling,

data analyses and writing of articles I, II, III & IV. Shared co-authorship

between J. M. Nielsen and M. Fusilli in article IV.

All accepted articles are reprinted with kind permissions from the publishers. The article

Nielsen et al. (2015) Meta-analysis of amino acid stable nitrogen isotope ratios for estimating

trophic position in marine organisms Vol 178, Issue 3, pp. 631-642, DOI 10.1007/s00442-

015-3305-7 was reprinted with permission of Springer. Nielsen & Winder (2015) Mar Ecol

Prog Ser 531:143–154 is reproduced in this thesis under license from the copyright holder.

The complete article from this source is not to be further copied separately from the thesis.

This restriction ends 5 years after the initial publication date.

TABLE OF CONTENTS

ABSTRACT v

LIST OF ARTICLES vii

TABLE OF CONTENTS viii

INTRODUCTION 9

Food web dynamics and nutritional flows 9

Diet tracing 10

Diet tracing using molecular methods 12

Stable isotope analysis for diet tracing 13

Compound specific stable isotopes 13

Zooplankton feeding ecology 16

OBJECTIVES 18

AIM AND MAIN FINDINGS 19

CONCLUSIONS AND FUTURE PERSPECTIVES 24

Tracer techniques 24

Zooplankton feeding dynamics 25

Future perspectives 27

ACKNOWLEDGEMENTS 31

REFERENCES 32

9

INTRODUCTION

Food web dynamics and nutritional flows

A primary field of research in ecology is the study of food webs (Elton

1927), which comprise a broad range of theoretical, mathematical and

empirical studies (Layman et al. 2015). Food webs are constructed of

multiple, potentially thousands of species interactions (Winemiller 1990),

which shape trophic nodes and flow of organic matter within a food web.

Certain species are often critical in shaping food web structure (Paine 1980),

yet these can vary due to variable predation and competition strengths

(Odum 1957, Dunne et al. 2002, Kratina et al. 2012). Despite being long

recognized as fundamental to ecology, an integrated assessment of food web

dynamics is still difficult due to the challenge in accurately assessing species

diet usage and trophic position within a natural system. Such information is

fundamental for categorizing food webs structure, complexity and energy

pathways.

Trophic linkages in food webs can be assessed in terms of energy flows

(Lindeman 1942), which also implies that the nutritional composition of

organisms are important in shaping trophic linkages. Nutrients, comprising

both atomic elements and organic molecules, provide the foundation for all

living organisms. Single elements are indispensable for organisms as they

provide the building blocks to the variety of organic compounds needed for

basic functioning. Elemental stoichiometry (i.e. the relative balance of

elements) thus provides a useful framework to assess how essential nutrients

flow between organisms (Sterner & Elser 2002).

While elements are indispensable, most organic compounds can be

synthesized or restructured by controlling enzymes. However, specific

compounds that cannot be synthesized de-novo by heterotrophic organisms

10

must be acquired through assimilation of dietary components and are

considered essential, such as highly unsaturated fatty acids (FA, Brett &

Müller-Navarra 1997) and certain amino acids (AA, Wu 2009). The

recognition that some molecules also act as essential compounds suggests

that understanding of dietary nutrition and its influence on consumer growth

goes beyond simple elemental stoichiometry (Müller-Navarra 2008). Often

more than one nutrient simultaneously governs species growth

(Raubenheimer & Simpson 2004, Wacker & Martin‐Creuzburg 2012).

While stoichiometry is often included in dietary models, few of them take

into account the role of nutritional quality of biochemical components

(Perhar et al. 2012). Fully understanding the role of different biochemical

compounds is crucial to better understand energy acquisition and nutritional

transfer between animals.

Diet tracing

Understanding nutritional flow across species trophic linkages relies on

suitable tracing methods to assess the relative importance and contribution of

dietary resources. In food web ecology a range of methods are used for

gaining insight on species food acquisition. These include visual or

molecular measures of stomach, faecal or gut content analyses (Pompanon et

al. 2012, Clare 2014) and stable isotope analysis of elements such as

nitrogen (N) and carbon (C) of bulk tissue (Post 2002, Boecklen et al. 2011)

or biochemical compounds (Bec et al. 2011, McMahon et al. 2011).

While the overarching aim among dietary measures is common in inferring

resource use of an organism, alternative measures (e.g. measure of

assimilation or ingestion) do not necessarily provide the same dietary

information, differing both quantitatively and qualitatively (Traugott et al.

2013). Consequently, the type of dietary information sought determines

11

which measure or combination of methods are most useful, along with

normal considerations of hypotheses, study design and the right spatial and

temporal scale. Ingestion is a measure of what is captured by the species and

thus provides information about what and potentially how many prey items

an organism has grazed upon. Such information can be obtained from visual

or molecular analysis of gut or stomach content. Assimilation can be defined

as the actual uptake of specific nutrients from the ingested diet which are

incorporated in a consumer’s tissue compartments. Assimilation, can for

example, be measured using stable isotope analysis. Thus, assimilation can

provide insight into which nutritional components are of importance for a

particular consumer.

Another important consideration in dietary analysis is the transformation that

the true diet components undergo before they are eventually measured either

in the stomach or as isotope composition of a consumers tissue. DNA

sequences of dietary items may be modified or partly broken by the time

they are measured in a consumer gut (King et al. 2008, Thomas et al. 2014),

and similarly the isotope ratio measured in a consumer’s tissue may not have

the same isotope composition of the initial dietary component (DeNiro &

Epstein 1981). Difficulty in accurately establishing such relationship

between diet and consumer is a continuous focal point in isotope

(McCutchan et al. 2003, McMahon et al. 2015a) and molecular (Thomas et

al. 2014) ecology. Regardless of the analytical method used, accurate

prediction of such transformation between original diet and measureable diet

is a key aspect in making accurate ecological predictions from dietary

analysis. For several dietary methods information on potential

transformation between consumer and diet is still scarce and usually

available from only a few species (Deagle et al. 2013, Hoen et al. 2014).

12

Diet tracing using molecular methods

Molecular tools such as DNA barcoding analyses of environmental DNA are

useful for gaining insight on species existence in the ambient water

(Thomsen et al. 2012), or dietary ingestion from gut or faecal content

(Pompanon et al. 2012). The use of DNA barcoding provides direct

information on dietary intake, including separation of individual prey items

to genera, taxa or species (Corse et al. 2010, Clare 2014). Diet analysis using

DNA methods rely on adequate reference libraries to identify the various

diet items, which can either be designed specifically by additional sampling

of potential dietary resources (García-Robledo et al. 2013), or alternatively

referenced from DNA databases, such as Genbank (Bohmann et al. 2011).

Depending on the molecular marker used, DNA barcoding may give explicit

information on both carnivory and herbivory (Clare 2014).

While detection of specific dietary items is a real strength of DNA

barcoding, quantifying dietary proportions can be more challenging.

Accuracy of diet quantification can be difficult both due to methodological

uncertainties or if prey organisms have different number of DNA copies of

genes per cell (Deagle et al. 2013), variation that is likely greater for species

belonging to different genera or families (Clare 2014). Detecting

cannibalism is difficult since consumer DNA is also normally present in the

diet sample (King et al. 2008). Despite these uncertainties the clear

advantages of direct diet information and the small amounts of material

needed for analysis make this a promising method for assessing species diet

intake. Molecular techniques seem particularly suitable for estimating

foraging patterns of small animals such as zooplankton (Hu et al. 2014)

which are often difficult to measure in their natural environment.

13

Stable isotope analysis for diet tracing

Stable isotopes of C and N are valuable tools for depicting both species

trophic information and diet assimilation (Peterson & Fry 1987, Post 2002).

Stable isotopes occur in nature as atoms of the same element having

different nuclear particle compositions, leading to small variations in mass.

The ratio between elements of stable isotope is described as:

δ(‰) = [(Rsample / Rstandard) -1] × 1000 (1)

where R = 13

C / 12

C or 15

N / 14

N of either bulk tissue or a specific compound.

The Rstandard is an international standard of atmospheric N2 for N or Pee Dee

belemnite for C. Alteration in isotope composition between a consumer and

its diet is known as trophic isotope discrimination (note: isotope

fractionation or enrichment are used interchangeably). Inferring species

trophic information or dietary assimilation relies on accurate knowledge of

the isotope discrimination factor. Stable isotope analysis have proven to be

very useful tools in ecology, however common for C and N of bulk tissue is

that isotope discrimination can vary. Variation in bulk isotope discrimination

is due to many co-occurring factors which can make interpretation difficult

without good ecological knowledge of the system or organism in question,

and we still need a better understanding of what shapes such variability

(Vanderklift & Ponsard 2003, Boecklen et al. 2011). Most stable isotope

work has focussed on bulk stable isotopes, which essentially is a measure of

a mix of multiple compounds, such as lipids, protein and carbohydrates.

Compound specific stable isotopes

A recent advance in stable isotopic techniques is the use of compound

specific stable isotope analysis (CSIA). Though sharing similar properties to

common bulk isotopes, C and N CSIA may provide additional resolution

when assessing species diet use and trophic position. CSIA of compounds

such as AAs provides an array of stable isotope measurements from a single

14

sample (~10-15 AAs). In principle such multiple tracers allow for separation

of an increasing number of consumer dietary resources, something that is a

common problem when using conventional bulk stable isotopes of only C

and N (Brett 2014). Multiple tracers also allow for source differentiation

using relative isotope ratios instead of absolute values (Larsen et al. 2009,

McCarthy et al. 2013), which may eliminate some of the uncertainties

associated with natural system variability when resources’ absolute isotope

composition are temporally or spatially dynamic. It has also been proposed

that multiple AA values can improve precision of trophic position estimation

(Sherwood et al. 2011, Décima et al. 2013, Nielsen et al. 2015). However, to

fully utilize multiple tracers more information on their isotope discrimination

patterns is needed.

Application of AA CSIA of

N isotopic composition is

being increasingly used to

infer trophic information of

both aquatic (Chikaraishi et

al. 2009, Bradley et al. 2015,

Nielsen et al. 2015) and

terrestrial species

(Chikaraishi et al. 2014). A

primary advantage of δ15

N

AA-CSIA is the dual

information from trophic

AAs, which encode

information about a species’

trophic position through

predictable 15

N trophic

discrimination, and source

Fig. 1

Change in consumer isotope ratios of

trophic (red) and source AA (blue) with

trophic position. The source AA isotope

ratio of all consumers on the food web

reflects the primary producers at the base

of the food web. Modified from

Chikaraishi et al. (2009).

15

AAs, which retain the N isotope composition of the primary producers that

synthesized them (Fig 1, McClelland & Montoya 2002, Popp et al. 2007,

Chikaraishi et al. 2009). Since N isotope discrimination can occur for several

essential AAs due to biochemical reworking of the amino group (i.e.

cleavage of C-N bonds), AAs are grouped into trophic and source AAs based

on the degree of N isotope discrimination. The δ15

N values of source AAs in

consumers provide a measurement in a consumer’s tissue of the nitrogenous

nutrients assimilated at the base of the food web (McClelland & Montoya

2002, Chikaraishi et al. 2009), something that can be difficult to accurately

measure when using bulk isotope composition.

Contrary to N isotope ratios, δ13

C AA values are commonly grouped into

non-essential (non-E-AA) and essential (E-AA), respectively, based on

consumer biosynthesis capabilities (Wu 2009). The δ13

C values E-AAs

which cannot be synthesized de-novo by heterotrophs have proven to be

effective tracers in natural systems due to clear differentiation in relative

δ13

C ratios between different types of primary producers (Larsen et al. 2013),

negligible trophic discrimination (Howland et al. 2003, McMahon et al.

2010), and consistency across changing environmental conditions (Larsen et

al. 2015). Differences in δ13

C E-AA values of aquatic invertebrate

consumers have previously been useful in distinguishing between resource

use of marine, terrestrial (Ellis et al. 2014, Vokhshoori et al. 2014) or marsh

primary producers (Fantle et al. 1999). Ecologists are continuing to explore

new ways to apply AA-CSIA to answer useful biological questions, yet an

important knowledge gap is what shapes variability in isotope trophic

discrimination in individual AAs and among different types of consumers or

diet sources.

Isotope discrimination can vary with the nutritional quality of assimilated

dietary components (Robbins et al. 2010, Ventura & Catalan 2010,

16

McMahon et al. 2015b). Previous studies have shown a clear relationship

between protein amounts and the isotope discrimination of both δ15

N and

δ13

C values (Gaye-Siessegger et al. 2004, Bloomfield et al. 2011).

Zooplankton bulk isotope values may also vary due to different nutritional

quality of the prey (Matthews & Mazumder 2005, Ventura & Catalan 2010),

while different dietary molecules can also vary in isotope composition

(Degens 1969, Kürten et al. 2013). Interestingly, N AA isotope

discrimination seem to vary both positively (Chikaraishi et al. 2015) and

negatively (Bloomfield et al. 2011, McMahon et al. 2015b) with diet

nutritional quality, highlighting the apparent complexity between animal

physiology and isotope incorporation and there is still much scope for

improved understanding of how isotopes and animal physiology are

interlinked.

Zooplankton feeding ecology

Zooplankton species are dominant intermediate species in aquatic

ecosystems and important prey items for many fish species (Stibor et al.

2004) due to their high abundance and high nutrient content (Beaugrand

2003). Yet, integrated assessment of how zooplankton species’ dynamics

influence food web energy flow remains difficult due to continuous temporal

and spatial environmental changes (Winder & Schindler 2004) and high

species complexity (Sprules & Bowerman 1988). The limited biosynthesis

capabilities of heterotrophs make the zooplankton-phytoplankton species

link especially influential for nutritional fluxes since certain organic

molecules must be taken up from the diet.

Optimal foraging theory predicts that species will target food items which

provide the highest nutritional gain relative to the costs associated with

foraging (Schoener 1971, Sih & Christensen 2001). Which dietary resource

provides the best choice for a zooplankton consumer differs among animals

17

(Sprules & Bowerman 1988). Zooplankton feed on a range of dietary

resources (Kleppel 1993), but target specific prey items which are

nutritionally rich (Peters et al. 2006) or of a suitable size for capture

(Berggreen et al. 1988, Katechakis et al. 2004). Zooplankton feeding likely

fluctuate throughout the season dependent on the availability of specific

dietary sources (Gentsch et al. 2008, Kürten et al. 2013) and the individual

organisms’ nutritional requirements (Mayzaud 1976, Villar-Argaiz et al.

2002).

Many mesozooplankton (grazer size ~ 0.2-20 mm) can also feed on protists

which provide important nutritional components (Boechat & Adrian 2006,

Mitra et al. 2013). Furthermore, the role of microzooplankton (grazer size <

0.2mm) as trophic upgraders (i.e. packaging and thus enhancing the

biochemical constituents of microalgae) and their ability to incorporate

bacterial C in aquatic food webs is likely more important than often

appreciated (Landry & Calbet 2004). The degree of omnivorous feeding in

zooplankton likely plays an important role in energy transfer in aquatic

systems and variation in feeding behaviour may alter food web structure

substantially (Sprules & Bowerman 1988, Kratina et al. 2012). The non-

static relationship between consumers and their diet assemblage (Grey et al.

2001, Nielsen & Winder 2015) makes accurate estimation of feeding

behaviour of zooplankton in their ambient environment critical for

understanding dynamics of energy flows in pelagic ecosystems (Dickman et

al. 2008). However, zooplankton diet resources are often difficult to measure

in natural environments and factors influencing zooplankton foraging and

seasonal feeding patterns are still not well resolved.

18

OBJECTIVES

Accurate estimation of energy and nutrient transfer between species is an

important part of categorizing food web dynamics. To measure such

properties, reliable empirical methods which trace nutrient fluxes are

essential. Dietary tracers, such as stable isotopes and molecular tools, have

greatly improved our ability to assess such energy flows between species.

Yet, we are still gaining new insights into how these tools can advance our

knowledge on natural food webs. Therefore, through an integrated research

approach, I studied the use of compound specific stable isotopes for

assessing predator-prey interactions, and applied molecular and isotope

tracer methods to categorize energy flow in natural aquatic ecosystems, with

a particular focus on the species links between phytoplankton-zooplankton.

Part 1: Investigation into the variability in AA stable isotope ratios across

trophic level and consumer type, assessed through a literature synthesis and

meta-analysis (article I) and a controlled feeding experiment (article II).

Part 2: Understanding predator-prey interactions and energy transfer in

aquatic ecosystems and across seasons, focusing on the link between

phytoplankton and zooplankton, using different tracer methods: stable

isotope analysis (article III) and molecular techniques (article IV).

19

AIM AND MAIN FINDINGS

Article I: Meta-analysis of amino acid stable nitrogen isotope ratios for

estimating trophic position in marine organisms

In this article, we compiled the first meta-analysis of δ15

N AA values from

measurements of 359 marine species covering 4 trophic levels. We

compared trophic position estimation using δ15

N AA values to previous

literature estimates from gut or stomach contents, and asked the following

questions:

1. Are N trophic discrimination factors in different AAs constant across

trophic position?

2. Does animal feeding ecology and form of N excretion affect the N trophic

discrimination factor?

3. Which trophic and source AA δ15

N ratios are most reliable for trophic

position estimation, and can estimates which incorporate multiple trophic

and source AA δ15

N values provide more precise trophic position?

Our meta-analysis confirmed the applicability of using AA δ15

N ratios as a

broad-scale tool to predict trophic position of marine organisms at all levels

of the food web. Within a given species, AA isotope ratios showed high co-

variation, supporting the use of multiple AAs to predict consumer trophic

position. Specifically, six trophic AAs all showed similar magnitude in

trophic isotope discrimination among individual measurements suggesting

ability to discriminate between trophic levels. Consistently low isotope

discrimination in two source AAs showed that these compounds reflect the

isotope ratios of primary producers at the base of the food web. Our findings

suggest that the isotope composition of these compounds can be combined to

retrieve species trophic position estimates.

20

Using a bootstrap approach we showed that in principle the N isotope

composition from an increasing number of source and/or trophic AAs

improved accuracy and precision in trophic position estimation. Our

modelling approach also showed that both the absolute value of the trophic

discrimination factor and uncertainty of this value is the most influential

source of error for trophic position estimation, emphasizing the importance

of applying accurate trophic discrimination factors to avoid under or

underestimation of species trophic position.

Another key finding of our meta-analysis was that organism feeding ecology

influences N trophic discrimination, with higher discrimination for

herbivores compared to omnivores and carnivores. N isotope trophic

discrimination was also lower for animals excreting urea compared to

ammonium. These findings suggest that researchers should consider using

group specific trophic discrimination values to improve accuracy in species

trophic position predictions.

Article II: Amino acid stable isotope discrimination in diverse aquatic

food chains

Here we studied multiple artificial freshwater food chains with various diet

combinations, covering trophic levels 1-3 and measured bulk tissue and AAs

isotope discrimination in C and N. We used model species of Daphnia

(Daphnia magna), fish (Poecilia sp.) and shrimp (Neocaridina heteropoda)

to represent common aquatic consumers, reared on a basal resource of

commercial fish pellet.

We estimated 1) N isotope incorporation rates of individual trophic AAs in a

consumer (shrimp) during a diet shift, and 2) C and N discrimination for

individual AA and bulk tissue, across trophic levels and resource type, and

21

assessed 3) if consumer isotope discrimination is influenced by nutritional

quality of the diet.

We found consistently low C isotope discrimination in E-AAs across two

trophic levels, confirming that these compounds are reliable dietary tracers.

Relatively high N isotopic discrimination was present in most trophic AAs,

while 15

N trophic discrimination remained relatively low in most source AAs

within feeding treatments. However, N isotope trophic discrimination in

most trophic AAs also varied among feeding treatments, including

consumers fed the same basal resource. For the majority of trophic AAs we

accredited this variation in N isotope discrimination to variable diet

imbalance, and we suggest that a consumer’s physiological adjustment in

response to variable food qualities can influence the magnitude of N isotope

trophic discrimination. Similar to our findings in article I, the laboratory data

indicated that N trophic discrimination can vary and that group or taxa

specific trophic discrimination factors are likely to provide improved trophic

position estimation.

Article III: Seasonal dynamics of zooplankton resource use revealed by

carbon amino acid stable isotope values

Here we used analysis of E-AA δ13

C ratios in combination with bulk δ13

C

and δ15

N values to study seasonal dynamics in resource use of a widespread

copepod consumer (Acartia spp.) in the northern Baltic Proper.

Our results indicated that Acartia used a range of dietary phytoplankton

groups, including dinoflagellates, cryptophytes, diatoms, prasinophytes and

cyanobacteria. However, the seasonal deviations between Acartia and seston

δ13

C E-AA values suggested that Acartia does not feed opportunistically on

all available components but instead preferentially selects and assimilate

22

AAs from certain diet items. The degree of selective feeding seemed to vary

across season. The distinct differences between Acartia and seston δ13

C E-

AA values further indicate that inferring trophic information from, for

example, N isotope composition of consumers relative to seston can be

misleading and such inference should be carefully evaluated or assessed in

combination with additional data.

Low summer δ15

N values in both Acartia and seston indicated that

diazotroph cyanobacteria play an important role for Baltic Sea zooplankton

either through direct or indirect utilization, in concurrence with previous

observations (Rolff 2000). The fact that Acartia δ13

C E-AA values seemed to

encode seasonal variation putative dietary sources supported the application

of δ13

C E-AA values as a suitable tool to study consumer resource

assimilation.

Article IV: Marine zooplankton diet preferences across species, life

stages and seasons using DNA barcoding

Here we used DNA barcoding to detect zooplankton dietary ingestion in four

common Baltic Sea zooplankton species: the copepods Acartia spp.,

Eurytemora affinis, Temora longicornis and the marine cladoceran Evadne

nordmanni.

We assessed the available food resources in the water column and compared

it to the diet composition of the different zooplankton species, life stages and

seasons (summer, autumn). We hypothesized that resource intake and

associated foraging patterns within a zooplankton community were dynamic

and depended on consumer species and the available diet biomass

composition. Specifically, we considered two alternative scenarios: 1) High

prey biomass will lead to high dietary overlap between zooplankton species

23

as consumers can easily target preferred items, and, 2) Increased competition

for resources will lead to higher levels of opportunistic feeding to fulfil

nutrient requirements for growth, and thus also diet segregation among

different zooplankton consumers.

Our molecular analysis provided direct identification of the majority of

autotrophic and heterotrophic dietary resources commonly ingested by

zooplankton species. All zooplankton consumers seemed to feed on a

relatively broad range of diet items but they did not feed opportunistically on

all available food sources. We found that zooplankton consumers, both

copepods and cladocerans, generally overlapped considerably in diet intake.

All consumers, including marine cladocerans, fed carnivorous on ciliate

prey. Our study also indicated that larger nauplii and copepodites were

capable of feeding on most of the same prey as adult copepods.

As competition for resources seemed to change across season, zooplankton

consequently changed their foraging behaviour. Yet, the degree of selective

feeding was not constant across season. Specifically, we found higher

dietary overlap between zooplankton when dietary biomass was high

indicating more specialized feeding, as the case during summer while in

autumn increased competition resulted in lower dietary overlaps because

zooplankton consumers likely fed more opportunistically.

24

CONCLUSIONS AND FUTURE PERSPECTIVES

Tracer techniques

A key component when depicting the structure of food webs is the ability to

place individual organisms within a food web. The broad scale applicability

of employing δ15

N AA values for predicting species trophic position was

supported in both our meta-analysis (article I) and laboratory study (article

II), in consensus with previous studies (Chikaraishi et al. 2009, Bradley et al.

2015). Our findings also highlighted the variability in species N isotope

discrimination, similar to what has been shown in other studies (Dale et al.

2011, Décima et al. 2013, Vokhshoori & McCarthy 2014). The variability

noted in our studies (articles I & II) indicates that researchers should

carefully evaluate trophic discrimination factors and consider employing

consumer specific values depending on taxa, dietary use (McMahon et al.

2015b), feeding behaviour (Nielsen et al. 2015) or excretion mechanism

(Germain et al. 2013, McMahon et al. 2015a).

Another observation in our laboratory study was that isotope turnover times

vary among different AAs (Article II, Bradley et al. 2014). Temporal tracing

methods have been applied to retrieve insight on assimilation efficiencies of

specific FAs (Mayor et al. 2011) and to assess how organic compounds such

as FAs are accumulated and stored (Koussoroplis et al. 2013). Studies into

isotope turnover times of individual AAs could similarly be very informative

in studying the importance of specific AAs for species growth and

nutritional usage.

An interesting pattern in our laboratory study (article II) was the consistent

co-variation between bulk and AA δ13

C ratios, suggesting that tracer

methods can be combined to yield ecological information. Such combination

25

may enhance separation among an increasing number of dietary sources,

something that is often problematic when using isotope ratios from only 2 or

3 compounds (Brett 2014, Phillips et al. 2014). It also implies that previous

studies about variability in bulk stable isotope ratios can provide valuable

knowledge about potential variation in AA-CSIA. These findings suggest

that even if CSIA can provide increased resolution in disentangling species

trophic information or diet use, bulk isotope and CSIA should be considered

complementary methods, rather than prematurely dismissing the application

of bulk stable isotope analysis (Bowes & Thorp 2015), which has shown to

be a very valuable tool applicable for answering a range of ecological

questions (Post 2002, Boecklen et al. 2011, Layman et al. 2012).

The advantage of having combined measurement of bulk and CSIA was

informative when disentangling possible selective feeding from changes in

trophic position in Baltic Sea zooplankton (article III). Our data suggested

substantial selective feeding, as shown by the increasing deviations in C AA

isotope composition between seston and zooplankton consumers during the

growing season. Changes in the difference between δ15

N isotope ratios of

consuming zooplankton and seston have previously been used to infer

changing trophic position across season (Boersma et al. 2015). However, our

findings indicate that inferring trophic information from N isotope

composition of consumers relative to seston should be treated with prudence,

since differences in δ15

N isotope ratios between seston and zooplankton

consumers may be a consequence of differences in feeding selection rather

than a change in trophic level (El-Sabaawi et al. 2010).

Zooplankton feeding dynamics

In both our studies (articles III & IV) zooplankton were found to feed on a

relatively broad range of resources. However, zooplankton did not feed

26

opportunistically on all available diet but targeted specific food sources

including dinoflagellates, diatoms, cyanobacteria and ciliates (article IV).

Different dietary resources vary in their nutritional composition (Brown et

al. 1997) and thus complementary diet components are seemingly needed to

adequately fulfil zooplankton energetic and physiological requirements

(Kleppel 1993, Meunier et al. 2015). A common phenomenon in our study

was ingestion of diet items which were usually above ~10 μm (approximated

from size measures of the microscopy data) and usually consisting of good

nutritional value in terms of lipids (Galloway & Winder 2015) and proteins

(Vargas et al. 1998). Larger mobile prey such as many protists were also

preferred, likely due to their high energetic gain (Klein Breteler et al. 1999,

Veloza et al. 2006).

We found a clear depletion in the N isotope composition of both

zooplankton and seston (article III). Such seasonal change in δ15

N isotope

ratios have previously been noted during summer blooms of cyanobacteria in

the Baltic Sea (Rolff 2000, Karlson et al. 2015). A high abundance of

cyanobacteria was also present in our genetic dietary data and indicated

considerable zooplankton ingestion of these algae (article IV), in

concurrence with previous findings (Motwani & Gorokhova 2013).

Cyanobacteria are often considered a relatively poor food source in terms of

nutritionally rich FAs (Galloway & Winder 2015). However, cyanobacteria

including diazotroph species which often fuel N production in summer

(Adam et al. 2015), can contain substantial amounts of N (Vargas et al.

1998) and may act as an important N source used to build proteinaceous

tissue in aquatic organisms.

An advantage of the molecular data (article IV, 18S analysis) was the ability

to assess the degree of zooplankton carnivory. The capability of all

zooplankton, including marine cladocerans to feed on both autotroph and

27

heterotroph prey such as ciliates suggests that most adult copepods should be

categorized as omnivores (Paffenhöfer 1988, Sommer & Sommer 2004).

Mesozooplanktons ability to feed on both autotrophic and heterotrophic prey

influences the energy and nutritional flows among lower trophic levels

species in aquatic food webs. Increased omnivory may enhance food web

stability and persistence (Kratina et al. 2012) and microzooplankton can also

introduce new energy and nutrients from bacterial components not accessible

for mesozooplankton. However, since energy is lost at every trophic level

increased omnivory may also introduce a longer and less efficient trophic

chain. In article III no data was available on protist diet resources and the

proportion of ciliate ingestion measured from molecular methods (article IV)

does not provide quantitative prediction of trophic efficiencies of energy

transfer. Nonetheless, our findings showed that nutritional flows in the

planktonic food web channel primarily through an omnivory zooplankton

food chain, which include considerable amounts of ciliate prey.

Our genetic study (article IV) revealed a high overlap in resource use

between different consumer genera. Such diet overlaps also suggest that

indirect competition between zooplankton can be high, and during peak

abundance in summer, marine cladocerans (Kankaala 1983, Möllmann et al.

2002) may also compete substantially with copepods for available

heterotroph and autotroph resources in the Baltic Sea.

Future perspectives

Molecular diet analysis showed to be a promising tool to depict individual

diet items (article IV) and thus measure zooplankton ingestion in natural

environments. Our molecular data seemed promising in identifying specific

food items however, accurate quantification of specific diet resources was

more challenging, something which seem a common problem in molecular

diet analysis (Deagle et al. 2013). A sensible way to increase quantitative

28

estimates is improved insight into the amount of DNA a given algal species

contains per individual cell or amount of C. This can be estimated by

complementary laboratory experiments where a known quantity of mono-

cultured cells or organism tissue is measured for amount of DNA sequences

(Thomas et al. 2014). Such additional a priori information will be very

useful in establishing relative contributions of diet intake in field samples. A

similar approach also seems promising for employing in environmental

monitoring schemes where molecular assessment methods are used to

quantify species abundances.

Fundamental requirements for an accurate diet tracer are predictable and

preferably negligible trophic discrimination and consistent analytical

reproducibility, which was the case for both δ13

C values of bulk and E-AA

values in our feeding study (article II). Precise diet tracers also require

adequate separation of resource components. Such separation of diet sources

has been established among largely different primary producer groups

(Larsen et al. 2013). However, further improving the application of δ13

C E-

AA values for dietary tracing relies on elucidating two key issues which

have not yet been studied. First, resolving to what degree dietary sources can

be separated within a given primary producer domain, such as among either

strictly aquatic primary producers or terrestrial plants. Second, measuring the

turnover rates of individual E-AA stable isotope ratios, as this provides

information of the temporal incorporation of specific AAs.

Future development of appropriate statistical frameworks will be highly

valuable to improve application of CSIA and allow full exploitation of the

information embedded in multiple tracer data (Galloway et al. 2014,

Swanson et al. 2015). Diagnostic methods are also needed allowing

evaluation of when CSIA data and associated models provide ecologically

realistic results. Such diagnostic tools have proven to be valuable when

29

assessing 2 or 3 tracers (Smith et al. 2013, Brett 2014), and when dealing

with complex multivariate data good diagnostic tools are even more crucial.

So far relatively few studies have taken advantage of combining ingestion

(e.g. DNA barcoding) and assimilation (e.g. CSIA) measures (Traugott et al.

2013, Chiaradia et al. 2014). Assimilation of compounds relate to nutrient

absorption and thus to growth and reproduction of a specific consumer.

Stable isotope analysis is especially useful for categorizing such energy

flow. Ingestion provides information about the effect of the upper trophic

level on the lower trophic level. In principle different dietary methods should

allow differentiation between the two and thus may be helpful in measuring

energy flow, species interaction strengths and predation pressures. Yet, to

what extent such mechanisms are actually possible to differentiate in

complex natural systems remains to be tested.

Another way to potentially merge data from different types of dietary

methods is by using DNA diet information as prior information in isotope

mixing models (Chiaradia et al. 2014). Such a combination seems a

promising way to further improve the output of statistical frameworks

aiming at elucidating ecological processes. The advantages of both

molecular and stable isotope methods for diet analysis are many and as we

increase insight on the use of these dietary proxies, vast possibilities of

potential ecological applications are likely to surface.

It is evident that food quality and not only food quantity plays an important

role in food web processes and trophic interactions (Mitra & Flynn 2007,

Perhar et al. 2012). Using appropriate diet tracers we showed that

zooplankton continuously alters feeding behaviour in response to the

changing dietary assemblage. Seasonal changes in diet assimilation influence

the nutritional composition of zooplankton themselves and propagate to

30

higher trophic level predators (Möllmann et al. 2004), something that alters

nutritional flows and community structure of aquatic food webs (Winder &

Jassby 2011). If we are to gain further understanding on how energy

transfers vary, it is necessary to look not only at elemental stoichiometry but

also on specific biochemical requirements in primary producers and

zooplankton.

Assessing how nutritional flows will be influenced by both natural and

human mediated perturbations is an important future task. Recent climate

warming has been shown to cause notable changes to phytoplankton

distribution, species composition and abundance in both marine (Reid et al.

1998) and freshwater (Winder et al. 2012) systems. Also, future climate

change is expected to increase bacterial activity and abundance of small-

sized flagellates (Wohlers et al. 2009), potentially adding new energy

pathways to aquatic food chains. Improved insight of how energy and

nutrient is transferred through entire natural communities as well as

identifying key trophic pathways that are especially crucial for shaping food

web structures, should thus be a continued focal point in aquatic ecology.

31

ACKNOWLEDGEMENTS

First and foremost I would like to thank my supervisor Monika Winder for

continued support and guidance. A special thanks to my assistant supervisor

Sture Hansson as well as Alfred Burian, and past and present members of the

Winder Lab group for enlightening ecological discussions.

I sincerely thank Katja Reutervik, Matteo Fusilli, Anna-Lea Golz, Helena

Höglander, Stefan Svensson, Jennifer Griffiths, Linda Eggertsen, Maria

Eggertsen, Olle Hjerne, Isabell Klawonn, Ragnar Elmgren, David Angeler,

Peter Hambäck and Clare Bradshaw for valuable help in the field,

laboratory, and/or for constructive comments and discussions during

manuscript writing. Thank you to collaborators Brian Popp, Rodrigo

Esparza-Salas and Love Dahlén.

I am especially grateful to the scientific staff at Askö, Eddie Eriksson, Eva

Lindell, Carl-Magnus Wiltén, Susann Ericsson, Matthias Murphy and

Barbara Deutsch and the Swedish National Environmental Monitoring

Program group at Department of Ecology, Environment and Plant Sciences,

Stockholm University for continuous help, support and data. Finally I would

like to thank my family.

This thesis was funded by the German Science Foundation (DFG) under

project number WI 2726/2-1.

32

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