Zooplankton growth and trophic linkages: Implications for fish feeding conditions in the Baltic Sea

Towe Holmborn

ii

Doctoral Thesis in Marine Ecology, 2009

Towe Holmborn, towe@ecology.su.se

Department of Systems Ecology

Stockholm University

SE-106 91 Stockholm

Sweden

© Towe Holmborn, Stockholm 2009

ISBN 978-91-7155-908-1

Printed by US-AB, Stockholm, Sweden

Cover: Zooplankton, by T. Holmborn

iii

To my precious

iv

v

ABSTRACT

The aim of this Thesis was to improve our understanding and assessment of feeding

conditions for zooplanktivorous fish in the Baltic Sea.

We investigated (papers I, II) the usefulness of biochemical proxies for assessments of

growth and metabolic rates in the dominant Baltic copepod Acartia bifilosa. A predictive

model (paper I) for egg production rate (EPR), based on body size, RNA content, and water

temperature, was established using females of different geographical origin. This model

demonstrates the usefulness of RNA content as a proxy for growth in zooplankton and,

together with abundance data, it could be used to evaluate fish feeding conditions. Further

(paper II), using A. bifilosa exposed to a food gradient, we evaluated responses of

physiological rates and other biochemical proxies for growth and established correlations

between physiological and biochemical variables. EPR and ingestion rate were most

significantly correlated with RNA content. As assayed variables saturated at different food

concentrations, food availability may affect assessments of physiological rates using

proxies. In paper III, we explored the effect of high EPR and ingestion rate on astaxanthin

content in A. bifilosa. We found that the astaxanthin content decreased at high feeding

rates, most likely due to decreased assimilation efficiency. This may impact the quality of

zooplankton as prey.

The invasion of Cercopagis pengoi, a zooplanktivorous cladoceran, has altered the trophic

linkages in the Baltic Sea food web. In paper IV, we evaluated the feeding of

zooplanktivorous fish on C. pengoi and found that irrespective of size both herring and

sprat feed on it, with large herring being more selective. In turn, C. pengoi feeds mainly on

older copepods (paper V), which are acknowledged important in fish nutrition. These

results indicate that C. pengoi may compete with fish due to the diet overlap.

Keywords: AARS activity, biochemical markers, Clupea harengus, copepod physiology,

food web interactions, non-indigenous species, RNA-based indices, Sprattus sprattus,

stable isotopes.

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SAMMANFATTNING

Denna avhandling syftar till att förbättra analysmetoder för och utvärdering av

födoförhållanden för Östersjöns djurplanktonätande fisk (tex stömming och skarpsill). I

studie I och II undersökte vi om biomarkörer kan användas för att mäta tillväxt (I, II) och

metabolisk aktivitet (I) hos den vanliga hoppkräftan Acartia bifilosa. Vi fann att RNA

innehåll i hoppkräftan väl speglade äggproduktionen, oberoende av honornas geografiska

härkomst (I). En prediktiv modell baserad på RNA-innehåll, honans storlek och

vattentemperatur förklarade 51% av variationen i äggproduktion. Vidare (II) undersökte vi

fyra olika biomarkörers förmåga att prediktera äggproduktion, respiration, och födointag i

ett experiment med olika födotillgångar. Honans RNA-innehåll beskrev äggproduktionen

och födointaget bäst. Men, eftersom olika variabler nådde sitt maxvärde vid olika

födokoncentrationer, kan prediktering av fysiologisk aktivitet med hjälp av biomarkörer

(som i modellen i studie I) vara missvisande vid höga födokoncentrationer (som tex under

en algblomning). Förutom kvantiteten av djurplankton är kvaliteten av de samma en viktig

faktor för fiskens födoförhållanden. En viktig antioxidant heter astaxanthin och bildas

bland annat av hoppkräftor efter födointag av ”byggstenar” som finns i alger. I den tredje

studien (III) undersökte vi om födokoncentrationen påverkar mängden astaxantin i A.

bifilosa. Vi fann att även om produktionsstatus och födointag ökade med ökad

födokoncentration så ökade bara astaxantininnehållet till en viss gräns innan det minskade

igen. Kontentan är att vid höga födokoncentrationer produceras många hoppkräftor, men av

sämte kvalitet, med avseende på astaxantin. I de två sista arbetena (IV, V) fokuserade jag,

via fältstudier, på konsekvenser för det pelagiska ekosystemet av den nyligen introducerade

rovvattenloppan Cercopagis pengoi. Vi fann (IV) att de två vanligaste djurplanktonätande

fiskarna i Östersjön (skarpsill och strömming), oavsett ålder, åt C. pengoi. Därför, beroende

på om C. Pengoi äter samma djurplankton som dessa fiskar, eller om de föredrar mindre

byten, kan deras närvaro i Östersjön minska alternativt öka födotillgången för fisk. I papper

V undersökte vi därför vad C. pengoi äter. C. pengoi föredrog stora hoppkräftor men även

till viss mån hinnkräftor och mindre djurplankton (hjuldjur). Totalt sett utgjorde stora

hoppkräftor den stösta andelen föda. Resultaten tyder på att C. Pengoi kan bidra till ökad

födotillgång för fisk, eftersom de bättre utnyttjar de små djurplanktonen, men även till en

ökad risk för konkurrens med fisk, då de framförallt äter samma typ av föda.

Informationen i denna avhandling kan hjälpa oss att förstå den viktiga länken mellan

djurplankton och fisk i den fria vattenmassans födoväv. Den typen av information är

nödvändig för att på ett hållbart sätt kunna förvalta Östersjöns fiskbestånd samt för att

förstå och prediktera framtida påverkan på ekosystemet orsakat av tex övergödning,

klimatförändringar och nyintroducerade arter.

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CONTENTS

ABSTRACT v

SAMMANFATTNING (in Swedish) vi

CONTENTS 7

LIST OF ABBREVATIONS 8

PROSPECT OF THESIS 9

LIST OF PAPERS 10

AN INTRODUCTION TO THE STUDY SYSTEM 11

The study area – The Baltic Sea 11

Crustacean mesozooplankton in the Baltic proper 11

Zooplankton in the Baltic Sea pelagic food web 13

ASSESSING ZOOPLANKTON GROWTH AND METABOLIC ACTIVITY 14

Using biochemical macromolecules to assess growth and metabolic activity in zooplankton 16

Creating a model to assess in situ egg production rate (paper I) 17

Comparing usefulness of different biomarkers (paper II) 18

Applicability of RNA-based models for in situ growth assessments in the Baltic Sea 19

QUANTITY – QUALITY 20

Carotenoids in the food web 20

Factors affecting astaxanthin levels in copepods 21

Copepod astaxanthin content in relation to food concentration (paper III) 21

THE ROLE OF NON-INDIGENOUS ZOOPLANKTON IN THE BALTIC SEA

PELAGIC FOOD WEB 23

C. pengoi as a prey (paper IV) 23

C. pengoi as a predator (paper V) 24

The overall impact of cladoceran NIS in the Baltic Sea pelagic food web 26

CONCLUSIVE REMARKS AND FUTURE PERSPECTIVES 28

ACKNOWLEDGEMENTS 30

REFERENCES 32

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LIST OF ABBREVATIONS

AARS Aminoacyl-tRNA synthetases

EPR Egg production rate (eggs ind-1

day-1

)

NIS Non-indigenous species

PL Prosome length (mm)

RNA content RNA content in females (µg ind-1

)

RNA:DNA Ratio of RNA and DNA content

RNA:protein Ratio of RNA and protein content

spAARS Protein specific AARS activity (nmol PPi mg protein-1

h-1

)

T Temperature (°C)

YOY Young-of-the-year

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PROSPECT OF THESIS

The overall aim of my Thesis was to improve assessments of fish feeding conditions

in the Baltic Sea and to increase our understanding of the link between zooplankton

and fish. To do this, I conducted studies on (1) physiological and biochemical

determinants of quantity and quality of copepod production, and (2) effects of a non-

indigenous zooplankton species on the pelagic food web and fish feeding conditions

in the Baltic Sea. This was, more specifically, done by:

Exploring the possibilities of using biochemical proxies to assess growth

and metabolic status in copepods (papers I, II). This approach could, for

example, provide us with useful analytical tools for assessments of in situ

zooplankton growth and nutrition, which are necessary for evaluating fish

feeding conditions and to refine food web models.

Investigating the consequence of high feeding and production rates on the

quality of zooplankton as prey. The quality aspect in this study (paper III)

concerned the important antioxidant astaxanthin. Studies of this kind will

help us understand connections between quantity of zooplankton and its

quality as a prey for higher trophic levels, including fish. In the Baltic Sea,

this is relevant with respect to the eutrophication that has altered the

general productivity status but also to the natural seasonal variations in

productivity.

Investigating top-down and bottom-up effects of the non-indigenous

predatory cladoceran Cercopagis pengoi in the Baltic Sea pelagic food web

(papers IV, V). Studying trophic interactions of this recently introduced

species with native prey and predators is crucial for understanding and

predicting the impacts of this non-indigenous species on the Baltic Sea

pelagic food web and fish feeding conditions.

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LIST OF PAPERS

The Thesis is based on the following papers, referred to in the text by their Roman

numerals (published papers are reprinted with kind permissions of the publishers):

I Holmborn T, Gorokhova E (2008) Relationships between RNA content

and egg production rate in Acartia bifilosa (Copepoda, Calanoida) of

different spatial and temporal origin. Marine Biology 153(3): 483–491.

©Springer

Holmborn T, Gorokhova E (2008) Erratum to: Relationships between

RNA content and egg production rate in Acartia bifilosa (Copepoda,

Calanoida) of different spatial and temporal origin. Marine Biology

153(5): 1007–1008. ©Springer

II Holmborn T, Dahlgren K, Holeton C, Hogfors H, Gorokhova E.

Biochemical proxies for growth and metabolism in Acartia bifilosa

(Copepoda, Calanoida). Submitted to Limnology and Oceanography:

Methods. Conditionally accepted manuscript

III Holeton C, Lindell K, Holmborn T, Hogfors H, Gorokhova E (2009)

Decreased astaxanthin at high feeding rates in the calanoid copepod

Acartia bifilosa. Journal of Plankton Research 31(6): 661–668. ©Oxford

Journals

IV Gorokhova E, Fagerberg T, Hansson S (2004) Predation by herring

(Clupea harengus) and sprat (Sprattus sprattus) on Cercopagis pengoi in

a western Baltic Sea bay. ICES Journal of Marine Science 61(6): 959–

965. ©Oxford Journals

V Holliland P, Holmborn T, Gorokhova E. Assessing diet of the non-

indigenous predatory cladoceran Cercopagis pengoi using stable isotopes.

Manuscript

My contributions to the papers were:

I Participation in planning of the study, all fieldwork, all analysis and data

processing, main part of the writing.

II Participation in planning and execution of the experiment (responsible for the

egg production and egg viability analyses), most data processing, main part of

writing.

III Participation in planning and execution of the experiment (responsible for the

egg production and egg viability analyses), commenting on the manuscript.

IV Participation in planning of the study, all analysis, except the one of the

zooplankton community, most data processing, writing of the first draft.

V Participation in planning of the study, microscopic analysis of the zooplankton

community, commenting on the manuscript.

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AN INTRODUCTION TO THE STUDY SYSTEM

The study area – The Baltic Sea The Baltic Sea is one of the largest brackish water seas in the world. It has a natural

salinity gradient with higher salinities in the south (Jansson and Velner 1995), and

therefore, the organisms living in the Baltic Sea is a mixture of freshwater and

marine species. Compared to fully saline or fresh water areas, the species diversity is

very low in areas with a salinity of 6–7 (PSU, A. Remane, according to Ackefors

1965), a salinity that corresponds to the conditions of my study areas; coastal

regions of the northern Baltic proper (Fig. 1).

BALTIC SEA

SWEDEN

FINLAND

I-V

.. I

Figure 1. Schematic map of the Baltic Sea. The black dots indicate areas in the northern Baltic

proper used for field sampling that provided data for this Thesis. The Roman numerals denote

the paper(s) in question for the sampling site. For a more detailed description of the sampling

sites see the respective papers.

Crustacean mesozooplankton in the Baltic proper Zooplankton is a diverse group with respect to e.g., taxonomy, life history traits,

behavior, and size. The size group of zooplankton called mesozooplankton (0.2–2

mm) consists mainly of small crustaceans, such as copepods and cladocerans. They

constitute a major link between primary producers (e.g., planktonic algae) and

higher trophic levels (e.g., mysids and fish).

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The calanoid copepods are among the most abundant and productive zooplankton

groups in the Baltic proper (Ackefors 1971, Ackefors and Hernroth 1972). They are

generally present in the water column throughout the year being more numerous

during summer (Ackefors and Hernroth 1972). There are several species of calanoid

copepods abundant in the northern Baltic proper; however, in coastal areas, only a

few species are found in great numbers, e.g. Acartia bifilosa and Eurytemora affinis

(Hessle and Vallin 1934, Ackefors 1981). The copepods of genus Acartia are

broadly distributed around the world (Bradford 1976) and are numerically dominant

or co-dominant copepods in most estuaries (Turner 1981, and references therein).

Acartia are free-spawning, opportunistic omnivores (Castel 1981) that feed on

phytoplankton, detritus and small heterotrophs depending on availability (Turner

1984, and references therein).

A. bifilosa (paper I, II, III; Fig. 2a) is a dominant species in many European

estuaries (e.g., Irigoien and Castel 1995, Burdloff et al. 2002) and can also be found

elsewhere in the northern hemisphere (Razouls et al. 2005–2009). In the Baltic Sea,

it occurs in salinities between 4 and 8 (Hessle and Vallin 1934, Viitasalo et al. 1994)

and tolerates a wide range of temperatures. However, it is rarely found in cold

waters or below 50 m depth (Hessle and Vallin 1934). Therefore, in the Baltic Sea,

A. bifilosa is abundant in offshore areas but is far more numerous and important in

coastal areas (Hessle and Vallin 1934, Hernroth and Ackefors 1979, Ackefors 1981).

In fact, it has been suggested that A. bifilosa is the most important copepod in

coastal areas of the Baltic proper (Ackefors 1981) and it is also recognized as an

important prey for fish (Turner 1984, and references therein, Arrhenius 1996, Voss

et al. 2003).

The cladocerans found in the northern Baltic proper are usually only present in the

water column during the warm season and overwinter as diapause eggs (Ackefors

1971). However, during the warm season they can be found in large numbers

(Hessle and Vallin 1934, Ackefors 1981). In archipelago areas, Pleopsis

polyphemoides, Bosmina maritima, and Cercopagis pengoi are the most abundant

species (Hessle and Vallin 1934, Litvinchuk and Telesh 2006).

C. pengoi (paper IV, V; Fig. 2b) is a predatory cladoceran native to the Ponto-

Caspian region (Rivier 1998). It was probably introduced form the Black Sea

(Cristescu et al. 2001) via the Volga-Baltic waterway (Leppäkoski and Olenin

2000). In 1992, the first specimens were recorded in the Gulfs of Finland and Riga

(Ojaveer and Lumberg 1995, Krylov et al. 1999), and in 1997, the first specimen on

the Swedish coast was found (Stockholm archipelago and Gotland basin; Gorokhova

et al. 2000). It has since spread to the southern regions of the Baltic proper (Bielecka

et al. 2000, Litvinchuk and Telesh 2006, Olszewska 2006), and from the Baltic Sea

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to the North American Great Lakes (MacIsaac et al. 1999, Cristescu et al. 2001). In

the Baltic Sea, C. pengoi is especially abundant in coastal areas (Litvinchuk and

Telesh 2006) and it is generally more numerous in the upper part of the water

column (down to 10 m depth) than in deeper layers (Krylov et al. 1999), which may

indicate its preference for warmer temperatures.

Figure 2. Species in focus in this Thesis: a) Acartia bifilosa, adult (CVI) female, the model

organism in paper I, II, and III; b) Cercopagis pengoi, gamogenetic female (barb stage III)

carrying two diapause eggs, the species in focus in paper IV and V.

Zooplankton in the Baltic Sea pelagic food web Generally, the population dynamics of fish stocks depends on their reproductive

success, i.e. successful spawning and survival of embryos and larvae (Fogarty and

O´Brien 2009). The reproductive success, in turn, depends on a variety of abiotic

variables, but also on feeding conditions for larvae (Kalejs and Ojaveer 1989,

Ojaveer and Lehtonen 2001). Nearly all fish species, including piscivorous fish, feed

and depend on zooplankton in their early life, as larvae and/or juveniles. The major

zooplanktivorous fish in the Baltic Sea are sprat (Sprattus sprattus) which is a

holozooplanktivore, and herring (Clupea harengus), whose diet also includes

mysids, amphipods, and small fish (Rudstam et al. 1992, Thurow 1997).

Due to the central position that zooplankton have in the Baltic Sea pelagic food web,

studies on zooplankton production, in terms of both quality and quantity, would

facilitate assessments of energy flow through the pelagic ecosystem, increase our

understanding of pelagic interactions and enhance predictive capacity of fish stock

modeling. However, during 1990–2006, the importance of zooplankton dynamics

and production was somewhat ignored and zooplankton monitoring was excluded

from the national monitoring programs. Recently, however, as a result of drastic

changes in the reproductive success, population dynamics, and physical condition of

many commercially important fish species, such as herring, sprat, cod, pike and

perch, there has been a growing interest in the dynamics and production of

zooplankton.

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ASSESSING ZOOPLANKTON GROWTH AND

METABOLIC ACTIVITY

Production assessments in continuously reproducing wild populations (such as most

zooplankton; Winberg et al. 1971) includes sampling, identification and counting of

different life stages (Winberg et al. 1971, Runge and Roff 2000), and calculations

based on an estimated or measured growth rate. Consequently, the production

estimate is a function of the growth rate estimate. Although assessment of in situ

growth may help us understand food web structure and dynamics, few such studies

on zooplankton exist. The reason for this is partly the lack of reliable and convenient

methods.

Today, common methods to assess growth include temperature-dependent models,

weight-dependent models, physiological or laboratory-derived budget models,

cohort or artificial cohort weight increase or moulting rate, and (for e.g. copepods

and rotifers) egg production assessment (reviewed by Winberg et al. 1971, and

Runge and Roff 2000). All these methods have shortcomings and their accuracy and

reliability for in situ growth assessment are often questioned. The temperature and

weight dependent models are simplified empirical growth models based on

temperature and/or body weight. The major problems with these models therefore, is

that the independent variables are limited to temperature and/or body weight only,

while other variables, such as food quantity and quality (Kleppel 1993), salinity

(Calliari et al. 2006), and physiological condition (health; e.g., Fahmi and Hussain

2003), age/life stage; Calbet et al. 2000) also may affect zooplankton growth. The

physiological method, on the other hand, which relies on previously determined

relationships between growth and e.g. weight, temperature, food availability,

respiration, and assimilation for in situ extrapolation have other problems. The major

problem, as suggested by Huntley and Lopez (1992) and Huntley (1996) is difficulty

in parameterizing the impact of different variables in the combined physiological

equation. The cohort or artificial cohort weight increase and moulting rate methods

use the dynamics of a single stage, cohort or a whole population to establish weight

increase or moulting rate over time in field sampled specimens incubated in the

laboratory (Runge and Roff 2000). The same rationale is employed in the egg

production method, where egg production in adult females represents the growth

(Runge and Roff 2000). In juvenile copepods, growth consists of somatic growth

(i.e., increase in body-mass and building exoskeleton), while in adults the growth is

generative (i.e., formation of sexual products; Carlotti and Nival 1992). The egg

production method can therefore be used only for assessing production in adult

female copepods, while the cohort or artificial cohort weight increase or moulting

rate methods should be used to assess growth in juvenile life stages. However,

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assuming a constant specific growth over different life stages during non limiting

food conditions, as suggested by Sekiguchi et al. (1980) and tested by e.g. Berggren

et al. (1988), the egg production method can also be used to approximate juvenile

growth. This assumption and therefore applicability of the egg production method

for the copepod production assessment has been challenged by many physiologists

(e.g., Richardson and Verheye 1999, Calbet et al. 2000). The major drawbacks with

the egg production and moulting methods are related to the risk of introducing

artifacts due to handling of the animals and to the enormous amount of labor

inherent to the methods. Nevertheless, the egg production method is generally the

least laborious of these methods, and is probably the most employed method for

direct measurement of copepod growth in field and laboratory studies (Runge and

Roff 2000).

Although direct growth measurements are crucial for production assessments, other

physiological variables are also useful for interpreting the condition and production

status of zooplankton. For example, a number of variables of metabolic activity such

as the rate of which an individual ingest food, egests or excrete non-assimilative

food products, and respire, all give us clues about what might inhibit or enhance

growth (Blažka 1971, Fig. 3).

Ingestion Egestion

AssimilationRespiration

Juvenile growth

(somatic + moulting)

Adult growth

(generative)

Trophic

transfer

Energy

Excretion

Figure 3. Schematic figure of energy flow through a copepod. The shaded boxes represent

variables assayed in paper I and II of this Thesis.

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Of particular interest is the ingestion rate as it sets the upper limit for all

physiological processes including growth (Fig. 3). Although these metabolic

indicators are valuable for understanding key mechanisms of growth-related

responses, the assessment of these variables are by nature as laborious as direct

growth measurements and often difficult to employ in field studies. Therefore,

alternative, easy to employ, yet reliable, methods for direct assessment of in situ

growth and metabolic activity in zooplankton could facilitate the accuracy of

zooplankton production estimates. This, in turn, would improve our ability to

produce more data, which would allow for a better understanding of food web

interactions; a prerequisite for reliable predictions of ecological models including

those evaluating fish feeding conditions.

Using biochemical macromolecules to assess growth and

metabolic activity in zooplankton There is an increasing recognition that biochemical indices can provide a suitable

alternative and/or complement to existing in situ methods for assessment of growth

and physiological condition in zooplankton (Runge and Roff 2000). In general, use

of these indices to assess physiological performance, such as egg production,

somatic growth, ingestion, respiration, and stress, are based on key components of

synthetic and/or metabolic pathways. These pathways are usually either directly or

indirectly linked to cell growth and synthesis of important biological

macromolecules. During recent decades, as a result of significant progress in

analytical methods (Caldarone et al. 2006), analysis of bulk ribonucleic acid (RNA)

has been increasingly used to estimate growth in a variety of small animals,

including cladocerans ( Gorokhova and Kyle 2002, Vrede et al. 2002) and copepods

(Nakata et al. 1994, Wagner et al. 1998). In copepods, the RNA concentration has

proven to be a sensitive indicator of both reproductive (Nakata et al. 1994, Saiz et

al. 1998, Gorokhova 2003, papers I and II) and somatic growth (Wagner et al.

2001), as well as a measure of metabolic activity in response to nutritional

conditions (Wagner et al. 1998, Gorokhova et al. 2007, paper II), salinity stress

(Calliari et al. 2006) and toxic substances (Gardeström et al. 2006). Studies

employing RNA-based indices usually standardize the RNA content of an individual

to either its DNA or protein contents. The underlying rationale for using RNA-based

indices is that RNA concentration, primarily, is a function of ribosome number

correlating with protein synthesis and hence with growth rate (Buckley et al. 1999).

The amount of DNA, on the other hand, is considered semi-constant and may

therefore be an indicator of the cell number, which is a rough estimate of the body

size. The RNA:DNA ratio therefore indicates the amount of RNA per cell. The

RNA:protein ratio reflects the number of ribosomes per amount of protein and hence

the cytoplasmic ribosome concentration (Bremer and Dennis 1996). Moreover, a

17

wide range of enzymes, such as the metabolic enzymes trypsin, citrate synthase,

lactate dehydrogenase, phosphofructokinase, and a moulting enzyme, chitobiase,

have been suggested as proxies for somatic growth, and physiological condition in

crustaceans (Mayrand et al. 2000, Sastri and Roff 2000, Lemos et al. 2002, Cullen et

al. 2003). The rationale of using metabolic enzymes is that the activities of these

enzymes are linked to adenosine triphosphate (ATP) production, which is required

for elevated growth. The rationale for using the moulting enzyme activity, on the

other hand, is related to the moulting cycle as a part of somatic growth process in

arthropods. Furthermore, in recent years, another enzymatic indicator of

zooplankton growth based on the activity of the aminoacyl-tRNA synthetases

(AARS) has been introduced and applied for copepod growth analysis (Yebra et al.

2005, paper II). The AARS are a group of enzymes important in catalyzing a critical

step of protein synthesis; i.e. the activation and attachment of amino acids to the

tRNA (Ibba and Söll 2000).

Creating a model to assess in situ egg production rate (paper I)

To produce robust predictive models, relationships between growth/physiological

condition and biochemical indices must be tested and validated before they can be

used for growth assessments of wild populations under varying environmental

conditions. In paper I, we evaluated factors regulating the earlier proposed RNA–

egg production rate (EPR) relationship in Acartia bifilosa (Gorokhova 2003) and

developed a model for in situ EPR assessment. This was done by measuring EPR

and RNA content in individual adult females sampled at geographically distant areas

(station effect) during spring and summer (season effect). We also measured surface

water temperature (T) and prosome length (PL). Multiple linear regressions were

generated to create a model that predicts EPR from RNA content, PL, station,

season, and T. We found that PL, RNA content, and season explained 53% of the

variation in EPR, nearly half of which was explained by RNA content alone. The

significance of season indicates the importance of factors varying with season. If

RNA content and EPR saturate at different food concentrations (as later found in

paper II), it is reasonable to believe that the RNA–EPR relationship is also affected

by food availability and possibly also food quality. Therefore, effect of the season

variable found in paper I could be, at least partially, related to the differences in food

between the seasons. Another factor that varies with season is temperature. Since

surface water temperature, which is a continuous and more precise variable than

season, was measured in this study, the season variable was replaced by the

temperature variable. This replacement did not cause a substantial drop in

explanatory power of the model. The effect of spatial origin (station) on the RNA–

EPR relationship was never significant. This implies that it may be possible to use

this model on the entire Baltic population of A. bifilosa as long as all variables are

within the range of assayed variables in paper I.

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Comparing usefulness of different biomarkers (paper II)

Although many studies have suggested, developed and refined biochemical methods

for assessment of growth and physiological condition, the results are often

contradictory and intercalibrations are much needed (Runge and Roff 2000). In

paper II, using A. bifilosa as a model species, we evaluated the functional response

of EPR, egg viability, ingestion, and respiration rates and four different commonly

used biochemical proxies related to protein synthesis [individual RNA content,

RNA:DNA ratio, RNA:protein ratio, and protein specific AARS activity

(spAARS)], in response to a broad spectrum of food concentrations. Then, a

correlative approach was used to evaluate the linkages between the biochemical

indices and physiological rates and thus the predictive capacity of these indices for

the physiological variables. There were three main findings in this paper: First,

although all variables (except egg viability) increased with increasing food

concentration either linearly (spAARS) or hyperbolically (all other variables), there

were substantial differences in saturating food concentrations among the variables.

Therefore, applicability of biomarkers as proxies of physiological rates should be

restricted to the non-saturated phase of the functional response of either variable,

unless both variables saturate at similar food concentrations. This restriction may

have implications for assessing copepod physiological rates at high food availability,

as during algal blooms. Second, RNA content and RNA:protein ratio were the best

proxies of egg production and ingestion rates, while RNA:DNA ratio and spAARS

activity were less suitable. The superior predictability of RNA content and

RNA:protein ratio is in line with previous findings in this species (Gorokhova

2003). Overall, the best correlation was observed for ingestion rate and RNA

content, a relationship not previously described for this species. Earlier, egg

production was suggested to be a more sensitive indicator of nutritional quality than

RNA:DNA ratios in Acartia tonsa (Speekmann et al. 2006). The strong relationships

between RNA content and ingested food quantity (II) and between EPR and

ingested food quality (Speekmann et al. 2006) confirms the importance of food on

the EPR–RNA relationship as proposed above (page 17). Third, RNA:DNA ratio

and spAARS activity were non-correlating variables, which implies that they could

be used jointly as co-variables in multiple regression models to predict ingestion

rate. In theory, joint use of non-correlating biochemical indices could be beneficial

to improve the predictive ability of models for growth and physiological condition,

but this needs to be explored further. Therefore, by investigating other biochemical

indices theoretically related to growth and metabolic pathways, but uncorrelated to

protein synthesis (e.g., compounds or rates related to ATP or DNA synthesis), we

may find other co-variables that can be used in such combined models.

19

Applicability of RNA-based models for in situ growth assessments in

the Baltic Sea

To better understand production processes in the Baltic Sea, knowledge of factors

governing zooplankton production is a prerequisite. Therefore, development of

robust models for growth assessment in zooplankton is an important issue for many

zooplanktologists. The findings of paper I and II provide good evidence that

individual RNA content can be used to assess in situ EPR in the common Baltic

copepod A. bifilosa, but much work still needs to be done before we can use this

approach to assess growth of the entire zooplankton community. The EPR-method

assumes that growth rates are the same for all copepod developmental stages, an

assumption proved to be incorrect when available food is not equally suitable for

juveniles and adults (Richardson and Verheye 1999, Calbet et al. 2000). In the

calanoid copepod Centropages typicus, this was especially pronounced when

copepods were feeding on particles larger than 5 µm (Calbet et al. 2000). For

production assessment of the entire zooplankton community, the growth–RNA

relationship for each species and each developmental stage needs to be defined.

Nevertheless, the model(s) proposed in paper I can be used “as is” to give a rough

measure of the productivity status of the zooplankton community. Moreover, our

findings reported in papers I and II suggest that RNA-based models for growth

assessment of A. bifilosa could be used over larger geographical areas, as long as

abiotic (e.g., temperature and salinity) and biotic (e.g., body size, food availability,

and food quality) factors are accounted for. Therefore, robust models of this kind

may be implemented in diverse monitoring programs, for construction of

productivity maps of fish prey. However, further area of concern with respect to

copepod population recruitment is that the EPR model described in paper I does not

discriminate between viable or total EPR, which has crucial implications for

estimating population recruitment. Yet, as shown in paper II, the proportion of

viable eggs was generally high and not related to total EPR or RNA levels. Indeed,

in our further study, that evaluated how egg viability is reflected in individual RNA

content of field collected Acartia tonsa females, no significant effect of egg viability

on the EPR–RNA relationship was observed (Hogfors et al., in prep.). On the other

hand, as egg viability decrease has often been observed in copepods feeding on

diatoms (Ianora et al. 2003), while our copepods were fed with a green algae

(Tetraselmis suecica, paper II) or ambient summer plankton (Hogfors et al., in

prep.), this needs further experimental studies with other prey. In the Baltic Sea,

diatoms are very abundant during spring blooms and therefore, caution should be

taken when inferring recruitment from biochemical proxies during diatom blooms,

in line with the recommendation to restrict use of biochemical assays for growth

assessment to unsaturated feeding conditions (paper II).

20

QUANTITY – QUALITY

Not only the quantity of zooplankton (addressed in papers I and II), but also their

nutritional quality are essential for determining feeding conditions for higher trophic

levels, such as zooplanktivorous fish and, consecutively, predatory fish, humans,

and seabirds in the Baltic Sea. An altered nutritional quality for zooplanktivorous

fish can result from an altered zooplankton community composition, as different

species may have different macro- and micronutrient contents (Persson and Vrede

2006). However, it may also reflect alterations in nutritional quality of certain taxa.

Effects of nutritional deficiencies in fish diet have been addressed earlier by many

studies in different aquatic systems, including the Baltic Sea. For example, changes

in the diet composition, and thus altered nutritional value, have been suggested to be

responsible for the decrease in the weight-at-age of Baltic sprat and herring (e.g.,

Raid and Lankov 1995, Rönkkönen et al. 2004). Although it is not clear why some

prey species seem to enhance fish growth, it may be related to species specific

variations in C:N:P ratio (Walve and Larsson 1999), their ability to store fatty acids

(Persson and Vrede 2006) or other biochemical differences.

Carotenoids in the food web There are many aspects of food quality, besides the energetic value, and fatty acid

and amino acid composition; these aspects include quantities and composition of

other essential macromolecules, trace elements, and minerals. During the last

decades, carotenoid pigments have been recognized to play an important part of the

food quality in aquatic food webs (Pettersson and Lignell 1999, Liñán-Cabello et al.

2002, Vuori and Nikinmaa 2007). Apart from being pigments necessary for

photoprotection, carotenoids have also showed characteristics of being antioxidants

and vitamin A precursors (Schiedt et al. 1985, Liñán-Cabello et al. 2002). The

carotenoid astaxanthin is acknowledged to be one of the strongest naturally existing

antioxidants (Edge et al. 1997), and has proved to be an efficient protector against

UVA and UVB radiation (Davenport et al. 2004). It is abundant in many marine

animals, but seems to be particularly important for crustaceans and salmonids

(Matsuno 1989). In the Baltic Sea, failed reproduction in salmon (Salmo salar) has,

at least partly, been associated with an early mortality syndrome (M74). This disease

seems to be connected with low levels of astaxanthin (Amcoff et al. 1998) and

thiamine (Pettersson and Lignell 1999) in the eggs and fry of affected females. Low

thiamine levels in Baltic salmon have been suggested to indicate an oxidative stress

(Vuori and Nikinmaa 2007), a condition that was possible to cure with an

astaxanthin enriched diet (Nakano et al. 1995). The diet of Baltic Salmon was,

however, very similar before any signs of M74 was seen and during the period when

this symptom peaked (Hansson et al. 2001). This suggests that any dietary

21

difference may be related to the quality of the salmon prey (primarily sprat and

herring).

In nature, astaxanthin is almost exclusively synthesized by animals from carotenoid

precursors, mainly β-carotene and zetaxanthin (Katayama et al. 1973, Foss et al.

1987) that must be supplied to the animals. In the Baltic Sea pelagic ecosystem, as

in most marine pelagic systems, copepods are likely to be the major producers of

astaxanthin. The astaxanthin produced by copepods is transferred to the higher

trophic levels. Of particular interest, therefore, are factors affecting astaxanthin

content in copepods.

Factors affecting astaxanthin levels in copepods

As astaxanthin production in copepods is dependent on the availability of precursors

in the diet, some studies dealing with factors affecting astaxanthin content in

copepods have focused on diet effects (e.g., Andersson et al. 2003, Van Nieuwburg

et al. 2005). Andersson et al. (2003) found the astaxanthin content per copepod to

increase at higher phytoplankton biomass and diversity, while it decreased at lower

phytoplankton biomass or high phytoplankton biomass dominated by diatoms. Van

Nieuwburg et al. (2005) on the other hand found a decreased astaxanthin content in

copepods feeding in nutrient (NP or NPSi) enriched macrocosms as compared to the

natural concentrations and proposed the lack of need for copepod photoprotection in

the higher algal biomass (and therefore lower water transparency) to be a plausible

explanation for the latter result. Moreover, they found no difference in astaxanthin

content per copepod between the two enriched treatments, dominated by different

algae. Other studies have looked at the importance of algae in starved/fed

experiments and found a positive relationship between availability of algae and

copepod astaxanthin content (Davenport et al. 2004, Sommer et al. 2006). Also, as

astaxanthin is recognized as a strong antioxidant with photoprotective properties, the

effect of light on astaxanthin production has been investigated. These studies found

that light was not a prerequisite for astaxanthin production (Davenport et al. 2004,

Sommer et al. 2006), although it seemed to enhance the astaxanthin content in

copepods (Sommer et al. 2006). Therefore, it is proposed that the main function of

astaxanthin is to improve the antioxidant protection of storage lipids, also in

situations where photoprotection is not required (Sommer et al. 2006). If this is the

case, astaxanthin content should be more strongly regulated by food intake than by

light.

Copepod astaxanthin content in relation to food concentration (paper III)

Although no study has investigated the response curve of astaxanthin content to

elevated food concentrations, it is reasonable to believe that an increased food intake

will contribute to a higher supply of astaxanthin precursors, which will support

22

higher astaxanthin production, and thus a higher astaxanthin content, at least up to a

saturation point. In paper III, we used A. bifilosa as a model species, to investigate

the effect of feeding activity on individual astaxanthin levels. In adult female

copepods exposed to a broad range of food concentrations, ingestion rate and EPR

along with astaxanthin content were measured. While both EPR and ingestion rate

increased with increasing food concentration, as expected, a different response was

observed for the astaxanthin content: It increased in concert with increasing food

concentrations up to 150 µg C L-1

, however at the two highest food concentrations

(600 and 1200 µg C L-1

), the astaxanthin content dropped significantly and became

similar to the concentrations observed in starved individuals. The decrease was

significant, even after adjustment for astaxanthin deposition in eggs, and coincided

with lower carbon assimilation efficiencies at these food concentrations. Although

the mechanisms of this decrease need further investigation, we propose that the

reduced astaxanthin accumulation resulted from reduced uptake of precursors,

because of decreased assimilation efficiency at the higher feeding rates. However,

irrespective of the mechanism, the ecological consequences of this phenomenon are

extremely relevant to eutrophication and fish feeding conditions. Indeed, in a

eutrophicated system, copepods of poor nutritional quality (with respect to

astaxanthin content) may reproduce at high rates and sustain high population

densities. Therefore, although this high zooplankton production may be considered

positive for the fish feeding conditions, the low quality of the prey may cause

astaxanthin deficiency in the predators. These findings also suggest ecological

effects on a seasonal scale as copepod quality (but not quantity) may decline during

algal blooms or in nutrient enriched environments with high productivity. In line

with this, in previous studies (Van Nieuwburg et al. 2005), reduced astaxanthin

content in copepods exposed to high food concentrations (and therefore low water

transparency) was proposed to be a result of a lowered demand for photoprotection.

An alternative explanation to the observed decrease could be the same physiological

mechanisms as those proposed in our study (III). However, further experiments

investigating these responses using other zooplankton and algal species, in mono

and mixed cultures, would greatly improve our understanding of factors affecting

qualitative aspects of food web interactions. Furthermore, it is plausible that other

important biochemical compounds for zooplankton and their predators (such as

other carotenoids, Liñán-Cabello et al. 2002, Sies and Stahl 2005; fatty acids, Kainz

et al. 2004; vitamins and microelements, Guillaume et al. 2001) may exhibit the

same response as seen for astaxanthin in paper III, thus exerting additional negative

effects on diet quality for the higher trophic levels. Therefore, response of other

essential elements and compounds to food concentration as well as ecological effect

of these responses need to be carefully investigated in controlled laboratory

experiments as well as in field surveys.

23

THE ROLE OF NON-INDIGENOUS ZOOPLANKTON

IN THE BALTIC SEA PELAGIC FOOD WEB

The Baltic Sea ecosystem, like any other aquatic ecosystem of low salinity, low

biodiversity and few specialized niches, is very vulnerable to introductions of non-

indigenous species, NIS (Carlton and Geller 1993). Today, about 30 NIS are

recorded in the Swedish waters of the Baltic Sea (Anon. 2009), including the

mesozooplankton species: Acartia tonsa (Copepoda), Cercopagis pengoi

(Cladocera), and Evadne anonyx (Cladocera) (Anon. 2009). In the eastern parts of

the Baltic Sea, the cladoceran Cornigerius maeoticus maeoticus, has also been

recently recorded (Rodionova et al. 2005). Since the biology of this species

resembles that of E. anonyx and C. pengoi (Rivier 1998), a further spread of C. m.

maeoticus in the Baltic Sea is likely.

The magnitude and mechanisms and thus the consequences of these introductions on

the native Baltic Sea food web are generally poorly understood. However, it could

be hypothesized that introduced predatory zooplankton species (such as C. pengoi,

E. anonyx and C. m. maeoticus; Rivier 1998) will alter the feeding conditions for

zooplanktivorous fish, either by competing with fish for food or by becoming an

additional prey for fish. In the Baltic Sea, most studies investigating the ecological

impact of these planktonic NIS have so far focused almost exclusively on C. pengoi,

as it was introduced much earlier than the other two cladocerans, and as it is still the

only planktonic NIS present in different sea areas every summer in substantial

quantities. However, it cannot be ruled out that the other newcomers will increase

their abundance with time and that already today their food web effects, albeit

perhaps less pronounced, are similar to those of C. pengoi, because of the

similarities in feeding biology of these species (Rivier 1998).

C. pengoi as a prey (paper IV) In the Gulf of Riga, Ojaveer and Lumberg (1995) have found herring (but not

young-of-the-year, YOY, herring) to feed on C. pengoi to a great extent. This has

also been reported from the northern Gulf of Finland, where C. pengoi became a

main food source from August to mid October for larger herring (Antsulevich and

Välipakka 2000). In the Gulf of Riga, other fish such as three-spined stickleback,

nine-spined stickleback and smelt were found to feed somewhat on C. pengoi, while

sprat, white bream and bleak were not (Ojaveer and Lumberg 1995, Ojaveer et al.

2004).

In paper IV, we investigated feeding of herring and sprat, the two most common

zooplanktivorous fish in the Baltic Sea, on C. pengoi. Contribution of C. pengoi to

24

fish diet was estimated by examining stomach content of fish from a coastal area of

the northern Baltic proper (Himmerfjärden Bay). The stomach content was then

compared to the ambient zooplankton community abundance and structure to

estimate prey selectivity. We found that both herring (5.2 to 25.2 cm) and sprat (5.7

to 11.6 cm) feed on C. pengoi. Overall, the proportion of fish with C. pengoi in the

stomach was 70 % and 61 % for sprat and herring, respectively. Moreover, herring

proved to become more selective for C. pengoi with increasing size. The fact that

YOY of both herring and sprat feed on C. pengoi, indicates that this prey is not too

large for fish as small as 5 cm long, as suggested earlier (Ojaveer and Lumberg

1995, Bushnoe et al. 2003). These results imply that trophic position of

zooplanktivorous fish has shifted up as a result of the invasion, a conclusion later

supported by Gorokhova et al. (2005). They used stable isotopes of C and N to

elucidate food web interactions between C. pengoi and potential prey and predators

and found that since the introduction of C. pengoi into the food web, the trophic

position of YOY herring has shifted from 2.6 to 3.4. Also, even smaller fish such as

the sand goby (1.4 cm; Ehrenberg 2008), sticklebacks (3.2 cm; Ojaveer et al. 2004),

and herring (4.1 cm; Ojaveer et al. 2004) have now been reported to feed on C.

pengoi. Therefore, it seems reasonable to believe that many other fish species at

most ages can utilize C. pengoi as a prey. Moreover, in addition to the impact that C.

pengoi may have on the diet of zooplanktivorous fish, it may also influence the diet

of other zooplanktivores, such as mysids. Indeed, Baltic mysids (Neomysis integer,

Mysis mixta, and M. relicta) were reported to feed on C. pengoi (Gorokhova 2006,

Gorokhova and Lehtiniemi 2007). This variety of predators indicates that C. pengoi

is well integrated into the Baltic Sea pelagic food web. However, whether this

actually contributes positively to the overall availability of prey for e.g. fish is

dependent on whether C. pengoi competes with fish for the same prey or not.

C. pengoi as a predator (paper V) As C. pengoi is zooplanktivorous, it is important to know its diet and prey

preferences, in order to evaluate its effects on the feeding conditions of

zooplanktivorous fish.

In the eastern Gulf of Finland, the impact of C. pengoi predation on the zooplankton

community has been evaluated and monitored (Telesh et al. 2001, Litvinchuk and

Telesh 2006). Their results, suggest that since the introduction of C. pengoi to the

area, its predation pressure on other zooplankton such as herbivores and suspension

feeders has increased exponentially. However, the data set used in these studies is

inadequate with respect to sampling frequency, and therefore modeling output is

questionable. In another study performed in the Gulf of Finland, correlative statistics

on field data suggested that predation by C. pengoi may affect seasonal dynamics

and/or spatial distributions of native cladocerans and rotifers (Põllumäe and Kotta

25

2007). In particular, Bosmina maritima tended to stay below the thermocline when

C. pengoi was abundant in surface waters. This was suggested by the authors to be a

predator avoidance response triggered by direct predation of C. pengoi on B.

maritima. Moreover, the authors suggested that a positive correlation observed

between abundances of B. maritima and C. pengoi may indicate a beneficial effect

of C. pengoi on B. maritima. This was proposed to be a result of C. pengoi preying

on other species that compete with B. maritima, such as Pleopsis polyphemoides and

Evadne nordmanni (as indicated by their negative correlations with C. pengoi).

Moreover, in the Gulf of Riga, the abundance and phenology of B. maritima has

changed after the introduction of C. pengoi (Ojaveer et al. 2004). The B. maritima

population has decreased and disappears from the zooplankton community several

weeks earlier than before the invasion (Ojaveer et al. 2004). A shift in the phenology

was also apparent in the copepod E. affinis who started to develop about three weeks

earlier following the introduction of C. pengoi (Ojaveer et al. 2004). Experimentally

determined feeding rates on E. affinis support the proposed negative impact of C.

pengoi on the E. affinis population (Lehtiniemi and Gorokhova 2008). As indicated

by the studies above, most research on C. pengoi feeding in the Baltic Sea pelagic

food web has employed correlative statistics on field data. When interpreting

correlative statistics it is important to remember that it only verifies co-varying

variables rather than expresses a dependency relationship. As there is a risk that two

variables, although independent of each other, co-vary as a result of another/or

several other external forces, the credibility of correlations to investigate

dependency relationships is fairly weak. Nevertheless, these studies suggest

plausible interactions and indicate what kinds of data are needed to verify these

interactions and increase our understanding of the impact of C. pengoi in the Baltic

Sea pelagic food web.

Stable isotope compositions can be used to assign trophic positions in food webs,

thus contributing to understanding energy flows and ecological relationships. This

approach employs relative concentrations of C and N isotopes in organisms to assess

dietary connections. The ratio between different isotopes of carbon and nitrogen

differs between organisms and their diets because of a slight selective retention of

the heavy isotope and excretion of the lighter isotope. As a result, organisms tend to

become enriched in heavy isotopes as compared with their food (Owens 1987). In

the Baltic Sea, this approach was used to evaluate food web interactions after the

introduction of C. pengoi (Gorokhova et al. 2005). It was concluded that

mesozooplankton (mainly copepodites) where the main prey of larger C. pengoi

while smaller C. pengoi fed equally much on microzooplankton (mainly rotifers and

nauplii) and mesozooplankton.

26

In paper V, we further investigated prey preferences of Cercopagis pengoi during

June–August using stable isotope analysis. The study was carried out in a coastal

area of the northern Baltic proper (Himmerfjärden Bay). Diet composition of C.

pengoi was determined from stable isotope signatures of the predator and possible

prey organisms, using two approaches: IsoSource mixing models (Phillips and

Gregg 2003) and temporal tracking of prey (Melville and Connolly 2003). In

parallel, zooplankton composition and abundance were analyzed to estimate

selectivity for different zooplankton prey. On a seasonal basis, the main contributors

to C. pengoi diet were copepods Acartia spp. and E. affinis, while importance of

cladocerans and microzooplankton was less apparent. C. pengoi behaves, overall, as

an opportunistic generalist predator, with a nutrition substantially overlapping that

of commercially important zooplanktivorous fish.

The overall impact of cladoceran NIS in the Baltic Sea

pelagic food web Earlier analysis on stable isotopes, conducted in the same area as our study (paper

V), suggested that C. pengoi feed, to a large extent, on copepods (i.e., the same food

items as YOY herring; Gorokhova et al. 2005). We found that copepods indeed

dominate in the diet of C. pengoi (paper V). Therefore, as not only YOY herring but

also other zooplanktivorous fish (e.g. sprat) at different age feed intensely on

copepods (e.g., Arrhenius 1996, Voss et al. 2003); this indicates a potential food

competition between C. pengoi and fish. However, C. pengoi appears to select

positively for podonids and also occasionally for microzooplankton, i.e. prey which

is less preferred by fish. Thus, during periods of low copepod abundance C. pengoi

may provide an energy link between lower trophic levels and fish.

It is clear that C. pengoi is well incorporated into the Baltic Sea pelagic food web,

where it acts as both an opportunistic predator (paper V) and a prey for fish (paper

IV) and mysids (Gorokhova 2006, Gorokhova and Lehtiniemi 2007), with many

complex interactions (Fig. 4). However, the overall effect of C. pengoi on fish

feeding conditions is still poorly understood. By integrating these results on fish

(paper IV) and C. pengoi (paper V) diets with field data on C. pengoi, fish and

zooplankton abundances collected in concert, the strength and outcome of the

interactions between these groups could be addressed in trophic interactions models.

Nevertheless, it is quite clear that C. pengoi alters the quantity and composition of

zooplankton available for zooplanktivores. It has, however, been suggested that

since C. pengoi, in the Baltic Sea, is only temporarily present in the water column

(from mid- to late summer) its overall impact will be limited to this period (Orlova

et al. 2006). Although this in principle might be true, it is important to acknowledge

that this short-term impact during summer may leave traces in the food web, which

27

may be significant all year round. This may be the case if the presence of C. pengoi

alters the feeding condition for fish and/or impacts the phenology of zooplankton, as

suggested for B. maritima (Ojaveer et al. 2004).

Zooplanktivorous fish Mysids

C. pengoi

Cladocerans (other

than C. pengoi)

Rotifers + copepod nauplii

Copepodites

1

IV

V

2

V

IV IV

V

IV

4

5

4

3

Figure 4. Pelagic food web interactions involving C. pengoi in the Baltic Sea. This schematic

food web is not complete and created mainly to visualize the findings of this Thesis. Arrows

represent confirmed interactions, and the number refers to the source of information; 1: Rivier

1998, 2: Gorokhova and Lehtiniemi 2007, 3: Arrhenius 1996, 4: Barz and Hirche 2009, 5:

Viherluoto and Viitasalo 2001. The Roman numerals represent papers in this Thesis.

The impact of the other newly introduced cladoceran species in the Baltic Sea, C. m.

maeoticus and E. anonyx remains to be investigated to see if these species will affect

the food web in similar ways, or if they will have other impacts. Moreover, the

unexpected (due to its lack of invasion history and previously described salinity

tolerances) successful establishment of E. anonyx into the Baltic Sea may indicate

that other common Ponto-Caspian onychopods (e.g. Podonevadne trigona,

Podonevadne camptonyx, Podonevadne angusta, Polyphemus exiguous and Evadne

prolongata) may exert an additional invasion threat towards the Baltic Sea (Panov et

al. 2007). Furthermore, due to prevailing climatic change, the establishment and

population abundance development of these species, originating from warmer

climate, may be reinforced in the future.

28

CONCLUSIVE REMARKS AND FUTURE

PERSPECTIVES

In the first two papers (I, II), we found that RNA content is a suitable biomarker for

assessing growth (expressed as EPR) in adult Acartia bifilosa females. The proposed

predictive model(s) for EPR assessment (I) could, therefore, as is, easily be

implemented in monitoring programs to assess zooplankton production, i.e. food

availability for zooplanktivores such as fish. However, to receive a more complete

picture of zooplankton availability for zooplanktivores, similar models should be

developed for other important zooplankton species and expanded to include juvenile

stages. Moreover, in order to use the model on a larger geographical scale (i.e,. the

entire Baltic Sea), the effect of salinity on the EPR–RNA relationship needs to be

tested and accounted for. This would greatly improve the usefulness of the model

because it would allow for productivity comparisons between different sub-areas of

the Baltic Sea. This information will help us understand variations in productivity

and recruitment success between different sub-populations of Baltic fish.

In paper III, we found that high food intake in copepods may lead to decreased

astaxanthin content. There may, however, be other factors, such as temperature,

salinity, light, and presence of algal or man-made toxins, which may further

modulate astaxanthin content in zooplankton. How, and to what extent, these factors

may affect astaxanthin content in copepods needs to be further explored. Moreover,

to fully understand factors affecting the quality of zooplankton as prey, the proposed

studies should also focus on other essential substances in a broader spectrum of

species.

Papers dealing with quantitative (I, II) and qualitative (III) aspects of copepod

production emphasize the importance of food quality and food quantity available for

the copepods. In the Baltic Sea, under conditions prevailing during a spring bloom

(i.e., high food concentrations and dominance of diatom species), the proposed

EPR–RNA model(s) for predicting A. bifilosa recruitment (paper I) may be less

reliable, due to the high food concentration (paper II) and the possible effects of

diatoms on egg viability (Ianora et al. 2003). Moreover, at high algal concentrations,

a decreased quality of the copepods in terms of astaxanthin concentration might be

possible (paper III). It is necessary to validate these findings in field as it will

impact the feeding conditions for zooplanktivorous fish.

Cercopagis pengoi is well incorporated into the Baltic Sea pelagic food web, acting

as both an opportunistic predator (paper V) and a prey for zooplanktivorous fish

(paper IV). Although the Baltic Sea holds a relatively small number of species,

29

which reduces the number of possible interactions, the trophic linkages of C. pengoi

are numerous (Fig. 4) and rather complex. Therefore, the overall effects of the C.

pengoi introduction on fish feeding conditions are likely to vary both seasonally and

regionally, depending on the food web structure. To evaluate these effects, long term

data for zooplankton, C. pengoi, and fish abundance/biomass that cover periods

before and after invasion should be integrated with the diet data (Holmborn et al., in

prep.).

In previous studies, correlation statistics on field data has been used extensively to

investigate food web interactions involving C. pengoi. We used fish stomach

analysis (paper IV) and stable isotope approach (paper V) to further understand

these interactions. Although IsoSource mixing models, especially combined with

temporal tracking of prey (as used in paper V), using stable isotope data provide

stronger proof of actual interactions between species in food webs than do

correlation studies, there are still some uncertainties with these methods. Therefore,

stomach analysis of C. pengoi using genetic markers of potential prey may be an

additional and even more reliable way to define and quantify these interactions.

The findings of this Thesis contribute to increase our understanding of processes

important for assessment and evaluation of fish feeding conditions and trophic links

between zooplankton and fish. This knowledge is crucial to make informed

ecosystem based management decisions concerning the Baltic Sea fisheries; an

approach that hopefully will improve and secure future harvest of Baltic sea-food.

Moreover, the knowledge gained in this Thesis will also help us identify and

understand future impact on the ecosystem caused by e.g. prevailing climate change,

eutrophication and introductions of non-indigenous species.

30

ACKNOWLEDGEMENTS

As Isaac Newton so neatly put it; “If I have seen further it is by standing on the

shoulders of giants”. In my case those giants are personified by my dear supervisors

Ass. Prof. Elena Gorokhova and Prof. Sture Hansson. Elena, thank you for countless

hours of interesting discussions during which you shared your vast knowledge with

me. Thanks also for using your sharp eye, excellent research skills, and scientific

curiosity to give constructive criticism and feedback on my work and for your ability

to always, and I mean always, be available for my questions and ideas, big or small.

Thank you also for facing everything with a smile, it made everything a lot easier!

It´s been great fun working with you and I hope the future will bring more

opportunities to work together. Sture, thank you for believing in me and for

introducing me to the department, and Elena, in the first place. Thank you also for

sharing your knowledge on fish ecology and for always encouraging and supporting

me through good and bad times.

Big hug to my amazing co-authors: Kristin Dahlgren (née Lindell), Elena

Gorokhova, Sture Hansson, Hedvig Hogfors, Claire Holeton, and Per Holliland.

Special thanks to the “copepod girls”; you made the most intense and productive

week of my PhD a great memory for life. I never knew it could be that fun to work

endless hours in the middle of the night with very tiny crustaceans! Thanks also to

Lisa Kosinkova, and Karolina Eriksson-Gonzales for various assistance in the

laboratory.

To all colleagues and friends at the Department of Systems Ecology – thank you for

creating a warm and productive atmosphere. Special thanks to my fellow PhD-

students. Thanks also to my former room-mates Lisa Almesjö and Marc Andersen

Borg for their patience over the years. I would also like to acknowledge the help I

have received from the staff at the chemistry laboratory. A special thanks to Berndt

Abrahamsson, Leif Lundgren, Stefan Svensson, Svante Nyberg, and Ulf Larsson,

who are all involved in the zooplankton monitoring. I would also like to

acknowledge the administrative staff that makes the whole research machine work

smoothly. Moreover, thanks to Börje Larsson, who caught all the fish for stomach

analysis, and Magnus Petersson, who kindly introduced me to methodological issues

concerning fish stomach analysis. Many thanks also to Jan-Olov Persson for

interesting discussions concerning statistics. Staff and colleagues at Tvärminne

Zoological Station (Finland) and Askö Field Station, thank you for everything! Also,

thanks to all friends and colleagues who challenged me in the weekly adventures

taking place in the floor-ball court. You gave me energy to tackle the work week! A

special thanks to Helena for introducing me to the happening of the week!

31

Jonas, you are the love of my life, my family, my friend and my steady rock. I know

that I will always have you there to pull me back up (or bring me back down) when

the road gets bumpy, and I know that by experience ;-). Thank you for your loving

support and for your way of always putting my scientific defeats in perspective .

Dear mum and dad, thank you for always supporting me and for encouraging me to

follow my own path. Theres, my beloved sister, thank you for all your love and

support. Thomas, Janice, Tilda, and Albin, thank you for making life happier and for

preparing me for an alternative career (SingStar® )! Thanks also to all other

friends and relatives who have supported me, you know who you are!

The work within this Thesis was kindly funded by: The Swedish Research Council

for Environment, Agricultural Sciences and Spatial Planning (Formas); Göte

Borgströms foundation; EU (through Swedish Board of Fisheries); the World Wide

Foundation (WWF); the Swedish Environmental Protection Agency

(Naturvårdsverket, through AquAliens and the Swedish National Monitoring

program); Walter and Andrée de Nottbecks foundation (Finland); Stockholm Marine

Research Centre (SMF); and the Royal Swedish Academy of Agriculture and

Forestry (KSLA).

32

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