Trophic complexit y of zooplankton –cyanobacteria
interactions in the Baltic Sea: Insights from
molecular diet anal ysis
Nisha H. Motwani
ii
Trophic complexity of zooplankton–cyanobacteria
interactions in the Baltic Sea:
Insights from molecular diet analysis
Nisha H. Motwani
Doctoral Thesis in Marine Ecology
Department of Ecology, Environment and Plant Sciences
Stockholm University
Stockholm 2015
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Doctoral Thesis in Marine Ecology, 2015
Department of Ecology, Environment and Plant Sciences
Stockholm University
SE-10691 Stockholm
Sweden.
©Nisha H. Motwani, Stockholm University 2015
ISBN 978-91-7649-118-8
Printed by Holmbergs, Malmö, Sweden 2015
Distributor: Department of Ecology, Environment and Plant Sciences
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To my parents,
Shri Wasu and Shrimati Pushpa Tolani
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ABSTRACT
Blooms of nitrogen fixing cyanobacteria (NFC) occur in many freshwater
and marine systems, including the Baltic Sea. By fixing dissolved nitrogen,
they circumvent general summer nitrogen limitation, while also generating a
supply of novel combined nitrogen for non-diazotrophic primary producers
and ultimately supporting secondary production. Elucidating trophic links
between primary consumers and NFC is essential for understanding role of
these blooms for secondary production. However, until recently, there was
no reliable method to quantify individual prey species for zooplankter feeding in situ. The development of PCR-based methods to detect prey-specific
DNA in the diet of consumers, including microscopic animals, allows identification and quantification of trophic linkages in the field.
Using molecular diet analysis in combination with egg production measurements, biochemical markers of growth and condition; and stable isotope
approach, we explored a possibility to determine (1) whether cyanobacteria
are grazed and assimilated by mesozooplankters (Papers I and II), (2) which
species/groups are particularly efficient consumers of cyanobacteria (Papers
II and III), and (3) how feeding on cyanobacteria affects zooplankton
growth and development (Paper I and III). Taken together, these laboratory
and field observations, provided evidence that NFC contribute to feeding and
reproduction of zooplankton during summer and create a favorable growth
environment for the copepod nauplii (Paper I). The favorable growth conditions for juvenile copepods observed during NFC blooms were hypothesized
to be mediated by picoplankton that take up bioavailable nitrogen exuded
from cyanobacterial cells. This hypothesis found support in Paper II that
provided quantitative estimates for the direct picocyanobacteria → mesozooplankton pathway, with highest weight-specific consumption observed in
nauplii. Further, using field observations on zooplankton and phytoplankton
development during a growth season in the northern Baltic proper, we found
that NFC nitrogen is assimilated and transferred to zooplankton via both
direct grazing and indirectly through grazing on small-sized phyto- and bacterioplankton (Paper III). Finally, these and other findings emphasizing the
importance of NFC for Baltic Sea secondary production during growth season were synthesized to show that diazotrophic nitrogen enters food webs
already at bloom initiation (Paper III) and is transferred via multiple pathways to pelagic and benthic food webs and, ultimately, to fish (Paper IV).
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CONTENTS
ABSTRACT
vii
LIST OF PAPERS
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1. INTRODUCTION
1.1. Baltic Sea cyanobacteria
1.2. Zooplankton grazers
1.3. Classical vs. Microbial food webs: the Baltic prospective
1.4. Transfer of diazotrophic nitrogen via picoplankton
1
1
2
3
4
2. SCOPE OF THE THESIS
2.1. Problem statement
2.2. Aim and objectives
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5
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3. STUDY AREA AND METHODS
3.1. The Baltic Sea and its pelagic food web
3.2. Molecular diet analysis: studying trophic interactions in situ
3.3. Molecular evidence for zooplankton feeding on cyanobacteria
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8
9
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4. SYNTHESIS OF RESULTS | MEETING THE OBJECTIVES
4.1. Cyanobacteria as a food source for zooplankton
4.2. Picoplankton: a non-existing food source for metazooplankton?
4.3. Effects of cyanobacteria on copepod growth
4.4. Diazotrophic cyanobacterial blooms support Baltic productivity
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13
14
16
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5. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
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6. ACKNOWLEDGEMENTS
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7. REFERENCES
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LIST OF PAPERS
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I. Hogfors H., Motwani N. H., Hajdu S., El-Shehawy R., Holmborn T.,
Vehmaa A., Engström-Öst J., Brutemark A. and Gorokhova E. (2014)
Bloom-forming cyanobacteria support copepod reproduction and development in the Baltic Sea. PLoS ONE 9 (11): e112692.
II. Motwani N. H. and Gorokhova E. (2013) Mesozooplankton grazing on
picocyanobacteria in the Baltic Sea as inferred from molecular diet
analysis. PLoS ONE 8 (11): e79230.
III. Motwani N. H., Duberg J., Svedén J. B. and Gorokhova E. Grazing on
nitrogen-fixing filamentous cyanobacteria and transfer of diazotrophic
nitrogen to zooplankton grazers in the open Baltic Sea. Manuscript
IV. Karlson A. M. L., Duberg J., Motwani N. H., Hogfors H., Klawonn I.,
Ploug H., Svedén J. B., Garbaras A., Sundelin B., Hajdu S., Larsson U.,
Elmgren R. and Gorokhova E. Nitrogen fixation by cyanobacteria
stimulates production in Baltic food-webs. Accepted in AMBIO
My contributions to the papers:
I. Performed qPCR analyses, contributed to writing and provided comments on the manuscript.
II. Responsible author; participated in experimental design, responsible for
most analyses and data processing.
III. Responsible author; participated in study planning, conducted field
sampling for zooplankton and picocyanobacteria, responsible for molecular data analysis and data processing.
IV. Provided zooplankton gut content data for Nodularia spumigena, contributed to writing and commented on the manuscript.
Additional papers which are not included in the Thesis
V. Gorokhova E., Lehtiniemi M. and Motwani N. H. (2013) Trade-offs
between predation risk and growth benefits in the copepod Eurytemora
affinis with contrasting pigmentation. PLoS ONE 8 (8): e71385.
VI. Engström-Öst J., Brutemark A., Vehmaa A., Motwani N. H. and Katajisto T. (2015) Consequences of a cyanobacteria bloom for copepod reix
production, mortality and sex ratio. Journal of Plankton Research, 1-11.
doi:10.1093/plankt/fbv004.
VII. Holliland P., Motwani N. H., Ogonowski M. and Gorokhova E.
Growth, reproduction and genetic variation: testing causes of variable
diel vertical migration in the copepod Eurytemora affinis. Manuscript
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Abbreviations
C:N
Chl a
DNA
HELCOM
ITS
mtDNA
N
N2
NFC
P
PC
PCR
POM
qPCR
RNA:DNA
rRNA
SIA
Ratio of carbon to nitrogen
Chlorophyll a
Deoxyribonucleic acid
Baltic Marine Environment Protection Commission
also known as Helsinki Commission
Internal transcribed spacer
Mitochondrial DNA
Nitrogen
Molecular dinitrogen
Nitrogen fixing cyanobacteria
Phosphorus
Picocyanobacteria
Polymerase chain reaction
Particulate organic matter
Quantitative real-time polymerase chain reaction
Ratio of RNA to DNA
Ribosomal ribonucleic acid
Stable isotope analysis
1. INTRODUCTION
1.1. Baltic Sea cyanobacteria
Cyanobacteria exist as single-celled, colonial and filamentous forms in pelagic and benthic habitats (Fig. 1). Nitrogen fixing cyanobacteria (NFC)
have occurred for some 7000 years in the Baltic Sea, since it became brackish (Bianchi et al. 2000). In aquatic systems, NFC play an important role in
nitrogen (N) cycle (Mulholland 2007), adding substantial amounts of fixed N
that support ecosystem net production (Karl et al. 1997, Larsson et al. 2001).
In the Baltic Sea, NFC blooms occur every summer. They are dominated by
Nodularia spumigena, Aphanizomenon flos-aquae and Dolichospermum spp.
(Stewart 1973, Mur et al. 1999, Fig. 1ab). Toxin production has been associated mostly with N. spumigena and Dolichospermum spp. (formerly Anabaena spp.) that produce hepatotoxins nodularins (Laamanen et al. 2001)
and microcystins (Halinen et al. 2007), respectively. While species composition of the filamentous diazotrophs remains the same, the ratio of toxic vs.
nontoxic cyanobacterial species observed in late summer has increased
(HELCOM 2003). Environmental factors, such as salinity and weather conditions, influence the occurrence of NFC blooms (Walsby et al. 1995). In the
Baltic Sea, these blooms are often considered to have increased in frequency
and magnitude in recent decades as a result of eutrophication (e.g. Kahru and
Elmgren 2014). Moreover, a further increase is expected to occur as a result
of the climate change (Neumann 2010, O’Neil et al. 2012).
Figure 1. The Baltic bloom forming diazotrophic filamentous cyanobacteria at
200× magnification and picocyanobacteria under green excitation at 1000× magnification. (a) Nodularia spumigena; (b) Aphanizomenon sp.; (c) Synechococcus spp.
(orange autofluorescence).
Photo: Helena Höglander
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The Baltic Sea picocyanobacteria (Fig. 1c) are dominated by Synechococcus-type strains (Sánchez-Baracaldo et al. 2008) that are also the major contributors to the picoplankton biomass here (Ohlendieck et al. 2000). In the
Himmerfjärden Bay, for example, picocyanobacteria contribute up to 25% to
the total autotrophic phytoplankton biomass (Larsson and Hagström 1982).
Although some freshwater chrococcoid cyanobacteria are capable of Nfixation (Stal 2009) as well as some heterotrophic bacteria in the Baltic Sea
(Farnelid et al. 2014, Farnelid et al. 2013), the dominating SynechococcusProchlorococcus clades are non-diazotrophic (Sánchez-Baracaldo et. al.
2014) and no evidence of N-fixation by Baltic picocyanobateria has been
found (Stal et al. 1999).
The evidence is accumulating that cyanobacterial blooms may have a more
important role in the Baltic food webs than previously assumed. In the Baltic
proper, zooplankton biomass peaks during the same period as that of cyanobacteria (Johansson et al. 2004). Organic matter and combined nitrogen produced by these cyanobacteria are incorporated by picoplankton and pelagic
organisms (Meyer-Harms et al. 1999, Ohlendieck et al. 2000, Rolff 2000,
Lesutiene et al. 2014), thus challenging the view that cyanobacteria do not
contribute to secondary production supporting fish nutrition. Direct grazing
on NFC has been observed in many grazers in the laboratory (Koski et al.
2002, Kozlowsky-Suzuki et al. 2003) and in the field (Meyer-Harms et al.
1999, Gorokhova 2009) studies. Picocyanobacteria are generally considered
to be inefficiently exploited by large copepods (Pace et al. 1990), whereas
juvenile copepods, rotifers and cladocerans may feed on this prey (Geller
and Müller 1981, Stockner and Shortreed 1989, Roff et al. 1995). The relative advantages and disadvantages of grazing on NFC and picocyanobacteria
in situ, however, have not been sufficiently evaluated.
1.2. Zooplankton grazers
Zooplankton is a diverse group with respect to taxonomy, life history traits,
and behavior. Copepods are the most abundant metazooplankton throughout
the world’s oceans, making up the majority of zooplankton biomass in the
epipelagic zone (Verity and Smetacek 1996). In the Baltic Sea, calanoid
copepods dominate throughout the year, both in abundance and biomass,
however cladocerans are also common and seasonally important, especially
in northern regions (Ackefors 1981), coastal zones and the Baltic proper
(Vuorinen et al. 2003 and references therein). Several species of calanoid
copepods are abundant in the northern Baltic proper; with Acartia bifilosa
and Eurytemora affinis being dominant (Ackefors 1981, Viitasalo et al.
1995). They are important prey of fish larvae (Arrhenius 1996) and zooplanktivorous fish, herring (Clupea harengus) and sprat (Sprattus sprattus),
2
which consume a significant proportion of the crustacean zooplankton
production (Rudstam et al. 1992, Thurow 1997).
1.3. Classical vs. Microbial food webs: the Baltic prospective
Energy supplied to higher trophic levels is transferred through two main
pathways (Fig. 2): the classical food chain, from larger phytoplankton to
metazooplankton, and the microbial food web, in which most of the nano(2–20 μm) and picoplankton (<2 μm) carbon is transferred to protists and
only its minor part is directly available to the higher trophic levels (Hagström et al. 1988, Wikner et al. 1990). The microbial food web is based on
picoplankton and small protozoans that are ubiquitous (Azam et al. 1983,
Barber 2007). The general concept is that picoplankton are linked to higher
trophic level by the cascading effects of mesozooplankton grazing on consumers of intermediate size fractions (Sherr and Sherr 1988, Wikner and
Hagström 1988).
The microbial food web, including autotrophs and heterotrophs in the picoand nanosize range, often accounts for a large portion of biomass production
and nutrient recycling in the pelagic zone (Jürgens et al. 1994). The main
predators of bacteria (Kuuppo-Leinikki 1990) and autotrophic picoplankton
(Kuosa 1991) are heterotrophic nanoflagellates, which are in turn grazed
upon by nearly all metazooplankters (Sanders et al. 1994, Jürgens et al.
1996), along with ciliates (Wiackowski et al. 1994, Wickham 1995). Adult
copepods generally feed upon prey of >5 µm (Mullin 1980), whereas nauplii
may capture smaller particles, since the morphology of the feeding appendages is different (Fernández 1979). Therefore, by feeding upon picoplankton,
they constitute a bridge between the microbial and classical food webs
(Azam et al. 1983). Thus, copepods (as a group) are the main trophic link
between primary producers and fish, alternating between the microbial and
classic food webs (Verity and Smetacek 1996, Calbet and Saiz 2005).
3
NFC
I, III
PC
II, III
IV
Figure 2. Conceptual drawing of the diazotrophic nitrogen pathway in the
pelagic food webs of the Baltic Sea. Red arrows indicate pathways addressed
explicitly in this Thesis, and Roman numbers refer to the Papers focusing on specific pathways. The classical food chain (green arrow) and microbial pathway
(navy blue arrow, the loop is encircled with dashed line) are commonly considered
in food web studies. Here, we emphasize direct feeding on NFC (as a part of the
classical chain) and picocyanobacteria (PC; as a part of microbial loop), which
facilitates transfer of diazotrophic N to grazers and, ultimately, to fish.
1.4. Transfer of diazotrophic nitrogen via picoplankton
During summer, the microbial food web receives substantial amounts of N
fixed by NFC and leaking out. Baltic picocyanobacteria that do not fix N but
use NH4+ released by NFC, were found to benefit from this extra supply of N
(Ohlendieck et al. 2000, Stal et al. 2003, Ploug et al. 2011). Although mesozooplankton grazing on single-celled picoplankton is generally considered to
be non-efficient (Pace et al. 1990, Kiørboe 2011), some field and experimental studies indicate that ingestion of unicellular picoautotrophs by copepod species does occur (Wilson and Steinberg 2010, Motwani and
Gorokhova 2013, Paper III) and that picoplankton can also be an important
source of new organic carbon for large zooplankton (Richardson and Jackson 2007). Thus, the grazer-picoplankton interactions are system-specific
and complicated by the trophic interactions and nutrient fluxes within the
microbial loop (Azam et al. 1983). If metazooplankton grazers, particularly
copepods, are capable to directly and efficiently utilize picoplankton, at least
as nauplii, this would facilitate a direct energy transfer from microbial producers to metazooplankton, surpassing the microbial loop.
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2. SCOPE OF THE THESIS
2.1. Problem statement
In the Baltic Sea, herbivorous marine grazers are often N-limited during
summer, hence, feeding on NFC may be an important adaptive trade-off.
Moreover, by utilizing production of NFCs, these grazers would enhance
secondary productivity of the system and food availability for zooplanktivores, including fish. This, however, would depend largely on the increased
production that might be derived from cyanobacterial addition to the diet.
This fueling of the Baltic productivity, is rarely recognized as most of the
experimental studies suggest that NFC production cannot be efficiently utilized by grazers, due to low palatability, toxicity and poor nutritional quality.
Consequently, the common assumption is that cyanobacterial production
does not efficiently propagate into food webs and does not contribute to fish
production. Elucidating the trophic links between primary consumers and
NFC is essential for testing this assumption and assessing contribution of
cyanobacterial production to secondary production in the Baltic Sea.
A key step towards understanding the utilization of a resource by a consumer
is to assess the consumption in environmentally relevant settings. Studying
zooplankton grazing is methodologically challenging, due to numerous constraints related to experimental design, artefacts and analytical limitations.
During the last decade, molecular diet analysis has gained a great potential in
trophic ecology as an approach for in situ gut content analysis. It is particularly advantageous for studying small grazers, such as mesozooplankton, for
which other methods, such as microscopic examination of the gut content or
pigment analysis, are suffering from major shortcomings.
2.2. Aim and objectives
The aim of my thesis was to understand whether mesozooplankton can efficiently utilize cyanobacterial production (NFC and picocyanobacteria) by
direct grazing. In the field and laboratory studies, I used molecular approach
in combination with other methods to determine:
(A) Whether filamentous cyanobacteria and picocyanobacteria are ingested
by mesozooplankton grazers?
Addressed by:
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(i) Analysis of presence and abundance of Nodularia spumigena and
Synechococcus spp. DNA in the guts of Baltic zooplankters using
qPCR (Papers I, II and III).
(B) Which species/groups are particularly efficient in grazing on picocyanobacteria and whether this prey is assimilated?
Addressed by:
(i) qPCR-based analysis of Synechococcus spp. DNA in the guts of
common Baltic zooplankters (copepods, cladocerans, and rotifers) and
(ii) feeding experiment with laboratory reared copepod Acartia tonsa
fed isotope-labelled picocyanobacteria (Paper II).
(C) Whether copepods ingest and assimilate diazotrophic cyanobacteria during cyanobacterial bloom?
Addressed by:
(i) qPCR-based analysis of Nodularia spumigena and Synechococcus spp. in zooplankton guts and
(ii) relating these measurements to changes in δ15N isotope signatures
of zooplankton determined by stable isotope analysis (SIA) (Paper III).
(D) What are the consequences of feeding on toxic filamentous cyanobacterium for fitness-related parameters in copepods?
Addressed by:
(i) qPCR-based analysis of Nodularia consumption by Acartia bifilosa and concurrent assessment of oxidative status, reproduction,
and juvenile development in a laboratory experiment,
(ii) short- and long-term field observations on egg production (Acartia tonsa) and copepod nauplii (Acartia spp. and Eurytemora affinis) growth status in relation to the abundance of NFC (Paper
I),
(iii) Growth indices (RNA:DNA ratio) of zooplankton in response to
grazing on N. spumigena and Synechococcus spp. and
(iv) C:N ratio in zooplankton in relation to N content in particulate
organic matter (POM) during cyanobacterial bloom (Paper III).
(E) What are the possible benefits of cyanobacteria and its consumption for
secondary production in the Baltic Sea?
Addressed by:
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(i) Synthesizing results from recent field and experimental studies
showing that diazotrophic N is efficiently assimilated and transferred in the food webs and reach consumers via direct grazing on
NFC and indirectly via other non-diazotrophic phytoplankton and
microbial consortia (Paper IV).
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3. STUDY AREA AND METHODS
3.1. The Baltic Sea and its pelagic food web
One of the major environmental issues for the Baltic Sea, which is one of the
largest brackish water body on Earth (Björck 1995), is eutrophication, due to
external nutrient loads from its large drainage basin area (Bergström and
Carlsson 1994) and limited water exchange with the North Sea. It has lower
biological diversity as compared to fully marine or freshwater systems,
which means fewer trophic linkages (Elmgren and Hill 1997). Since in the
beginning of the 1980s, the pelagic ecosystem has changed remarkably. The
decrease in salinity and overfishing has influenced the reproduction of cod,
leading to a significant decrease of cod stocks in the 1990s (Plikshs et al.
1993). The important food item in the diet of pelagic fishes is zooplankton,
which has also been affected by the changes of salinity causing, for example,
a decrease of large zooplankton in the Gulf of Finland (Lumberg and Ojaveer 1991) and in the Baltic proper (Flinkman et al. 1998). In marine pelagic
food webs, zooplankton plays an important role in energy transfer between
primary producers and pelagic fishes, and is thus a major factor affecting the
food web efficiency and fish production. Zooplankton, mysids (Mysis sp.),
herring and sprat are the key constituents in the pelagic marine food webs in
the Baltic Sea.
In the Baltic Proper, summer primary production is often limited by nitrogen
(Granéli et al. 1990), and N-fixation by the most important diazotrophic
cyanobacterial genera, such as Aphanizomenon and Nodularia, is fundamental to its productivity (Stal et al. 2003). NFC biomass is increasing towards
the end of July, when the grazing pressure on phytoplankton and predation
on zooplankton are highest (Kuosa 1991, Kononen et al. 1996; Fig. 3). Indeed, mesozooplankton abundance in the Baltic Sea peaks during the same
period as cyanobacteria (Johansson et al. 2004), with calanoid copepods
being the most important group, both in terms of the biomass (Ackefors
1981) and diets of zooplanktivorous fish (Hansson et al. 1990). One might
speculate that when other edible phytoplankton are scarce, zooplankton may
rely more on the abundant cyanobacteria, despite their inadequate biochemical composition and even toxicity. The summer metazooplankton community is dominated by copepods (Viitasalo et al. 1995), and fecal pellets produced by copepods are important as sedimenting material (Turner 2002).
Copepods may therefore play an important role in both transferring diazotrophic N to the upper trophic levels and the vertical flux of it to the benthic
communities (Verity and Smetacek 1996). Hence, it is important to understand the complexity of zooplankton–cyanobacteria interactions, if we are to
provide a reliable assessment of the fate of NFC production in the system for
8
conclusive arguments and recommendations for policy and management
strategies.
Figure 3. Seasonal development of (a) phytoplankton, including cyanobacteria,
(b) zooplankton and (c) food consumption by zooplanktivorous fish in the
northern Baltic proper (Paper IV). During June–September, cyanobacteria contribute substantially to the phytoplankton communities, which coincide with the
highest zooplankton stocks, and the highest food consumption by fish. The phytoplankton and zooplankton data are long-term means (1992–2011) for the Askö area
(station B1) and fish food consumption data are from Arrhenius and Hansson 1993.
3.2. Molecular diet analysis: studying trophic interactions in situ
Traditionally, invertebrate diets have been analyzed by either intrusive or
non-intrusive methods. The intrusive methods consists of in vitro bottle incubations that allowed calculation of feeding rates based on estimates of
disappeared prey from the media or its appearance in the guts or feces
(Paffenhöfer 1988), which are considered to be direct and simplest. The
main limitation associated with this methodology is what is known as “bottle
effects” (Roman and Rublee 1980), the term referring to variations in turbulence, light regime or interactions with the container walls experienced by
grazers, which may affect their feeding behavior compared to their natural
9
environment (Båmstedt et al. 2000). Feeding preferences were also studied
by non-intrusive methods based on microscopy or a marker analysis of
water, gut and fecal pellet contents (Øresland and Ward 1993, Billones et al.
1999, Fleddum et al. 2001, Kaarvedt et al. 2002). These techniques that
make use of various markers to recognize and quantify ingested prey include
pigment-based methods to measure chlorophyll and other pigments in the
guts (Kleppel et al. 1993, Buffan-Dubau et al. 1996), amino acids (Guisande
et al. 2002) or fatty acids (reviewed in Dalsgaard et al. 2003 and Lee et al.
2006). To determine relative contribution of different food sources to a consumer diet, SIA has been widely used (Sato et al. 2002, Gorokhova et al.
2005, Sommer et al. 2005). Of these methods, the most of what we know
about zooplankton feeding in the field is based on pigment (Chl a) analysis
of gut contents, because it is fast and inexpensive. However, our understanding of prey selectivity in these consumers is largely based on laboratory incubations with manipulated food mixtures.
The microscopic examination of gut content is the direct way of diet analysis, however it is laborious and often biased, because only prey with hard
parts capable of surviving ingestion are preferentially detected (Töbe et al.
2010). There are also sources of error involved with gut fluorescence measurements that can lead to underestimation of the pigment ingested and,
hence, the grazing impact (Pasternak et al. 2005, Descy et al. 1999). Moreover, the pigment analysis is obviously restricted to autotrophic prey items
that are pigmented. Thus, the method is limited to strict herbivores and suffers poor taxonomic resolution, limiting our ability to distinguish between
important prey items (Peterson and Dam 1996, Roman et al. 2000, Liu et al.
2005). Therefore, search for biomarkers that would allow such resolution is
important. One of such biomarkers is a DNA molecule. DNA-based techniques are valuable in tracking predator-prey interactions, because they do
not rely on the visually diagnostic remains. A major advantage of using prey
DNA as a marker in gut content analysis is that DNA extracted from the
entire body of a consumer, or, alternatively, its digestive organs or faeces are
used, without introducing any bias from laboratory incubation.
Over the last two decades, methods based on polymerase chain reaction
(PCR) have been used to track trophic interactions in food webs; however
most of these applications have been limited to terrestrial insects and mammals (reviewed in Symondson 2002, Sheppard and Harwood 2005 and references therein). In aquatic animals, one of the first PCR-based diet analysis
was the study by Asahida et al. (1997), who developed a method to identify
mitochondrial DNA (mtDNA) of predated stone flounder larvae and juveniles in the guts of sand shrimp using PCR. Up to date, the genetic techniques based on PCR amplification of DNA have been successfully applied
in marine vertebrates (Jarman et al. 2002, Jarman et al. 2006, Rosel and
Kocher 2002) and invertebrates (Blankenship and Yayanos 2005, Galluzzi et
10
al. 2005, Vestheim et al. 2005, Martin et al. 2006). In quantitative PCR
(qPCR), quantification is achieved by detection of a fluorescent dye that
accumulates in direct proportion to the yield of amplified products. This
method can both identify prey of interest and quantify its amount (Troedsson
et al. 2007, Nejstgaard et al. 2008, Simonelli et al. 2009, Engström-Öst et al.
2011). This approach is gaining attention because it is relatively simple and
inexpensive to design primer sets for PCR that target organisms at various
taxonomic levels and at various degree of degradation due to digestion.
There are several studies that have used PCR and qPCR for detecting prey
DNA and investigating the feeding behavior of calanoid copepods (Nejstgaard et al. 2003, Gorokhova and Engström-Öst 2009, Engström-Öst et al.
2011), appendicularians (Troedsson et al. 2007) and mysids (Gorokhova and
Lehtiniemi 2007, Gorokhova 2009).
Multicopy target regions are favored in PCR-based diet analysis, because
they are abundant in cells and hence are more likely to be detected than single copy genes (Zaidi et al. 1999). An alternative to the multicopy mitochondrial genome that has been used successfully in predation studies is the
nuclear 18S rRNA gene (Nejstgaard et al. 2003, Jarman et al. 2004, Passmore et al. 2006). The rRNA genes have a generally lower substitution rates
and are potential markers for designing group-specific primers. They are also
represented well for various phylogenetics groups in public databases. This
makes it possible to identify rRNA gene fragments to a desired level of taxonomic resolution. Moreover, internal transcribed spacer (ITS) regions have
been effectively used to look at fine-scale differences between closely related species (Hillis et al. 1996).
3.3. Molecular evidence for zooplankton feeding on cyanobacteria
With the PCR-based detection of cyanobacterial DNA in the gut of grazers,
N. spumigena was found to be a common food both for mysids (≈58%;
Gorokhova 2009) and copepods (nearly 100%; Gorokhova and EngströmÖst 2009). Moreover, this cyanobacterium is not only ingested but also digested by the mysids, as indicated by lower yield of cyanobacterial DNA in
mysid’ feces than in the stomach bolus (Gorokhova 2009). Common zooplankton, the calanoid copepods Eurytemora affinis (Engström-Öst et al.
2011) and Acartia bifilosa (Paper I) as well as cladoceran Bosmina maritima (Paper III), actively ingest N. spumigena (Fig. 4) even when alternative
food is present. Thus, zooplankton grazers feed on NFC, bypassing the microbial loop. This direct pathway is not unique to the Baltic Sea; qPCRbased analysis has shown that toxic cyanobacteria can contribute to the diet
of herbivorous zooplankton both during bloom (Sotton et al. 2014) and nonbloom (winter) conditions (Oberholster et al. 2006) also in freshwaters.
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A qPCR assay has been also developed for Synechococcus, a large picocyanobacteria clade that constitutes up to half the phytoplankton biomass in the
offshore Baltic Sea (Hajdu et al. 2007). With small size and high growth
rate, picocyanobacteria are most likely to benefit from the supply of fixed
combined N leaking from filamentous cyanobacteria (Ploug et al. 2011).
Efficient utilization of picocyanobacteria by multicellular grazers will enhance the effect of cyanobacteria on secondary production. All main zooplankton groups feed on Synechococcus, which may make up 8–10% of the
gut content in copepodites of Acartia and Eurytemora and 15–30% in the
smaller cladocerans, rotifers and copepod nauplii (Paper II). This direct
metazooplankton grazing on non-diazotrophic picoautotrophs, which efficiently utilize diazotroph exudates, is thus an important route of N and energy for primary consumers in summer.
Figure 4. Abundance of Nodularia spumigena (mean ± SD) in the stomach bolus of copepods (Paper IV). Acartia spp. (A) and Eurytemora affinis (E) measured
in the field (open symbols) and in feeding experiments (bars), as a function of the
concentration of the cyanobacterium.
12
4. SYNTHESIS OF RESULTS | MEETING THE OBJECTIVES
4.1. Cyanobacteria as a food source for zooplankton
Nitrogen is an essential major element for life, accounting for about 10% of
the dry weight in aquatic organisms and located mostly in proteins (Sterner
and Elser 2002). Herbivorous marine grazers are often N-limited, as animal
tissue has a greater proportion of N per unit weight than plant material
(Sterner and Elser 2002). From this point of view, NFCs, including Baltic
filamentous cyanobacteria, are an attractive food source as they generally
have higher N content than non-diazotrophic phytoplankton (Walve and
Larsson 2007). Using both nitrogen isotopic signals and cyanobacterial pigments as tracers, copepods in the northern Baltic have been found to assimilate cyanobacteria in measurable quantities (Meyer-Harms et al. 1999). Although NFC are considered inadequate food source, due to their toxic nature,
morphology (large colonies that are difficult to manage), and less than complete nutritional content (Bury et al. 1995, Wilson et al. 2006), there are
grazers that have adapted to feeding on cyanobacteria (Engström et al.
2000).
In Paper I, we explored linkages between Baltic Sea NFC and copepod recruitment in terms of the reproductive output and juvenile development. To
evaluate the connections at ecologically realistic settings, we conducted a set
of interrelated studies in the northern Baltic Sea as follows:
(1) summer field survey in a coastal area to investigate linkages between phytoplankton, reproduction and growth indices in Acartia
tonsa,
(2) feeding experiment to test effects of Nodularia spumigena on oxidative balance, reproduction and development in A. bifilosa and
(3) analysis of long term (1999–2009) data to reveal relationships between NFC and growth indices in nauplii of Acartia spp. and Eurytemora affinis.
In the summer field survey, we used a correlative approach to relate reproduction (as egg production and egg viability) of the copepod A. tonsa to phytoplankton biovolume and community structure, with specific focus on NFC.
In this data set, positive effects of N. spumigena on the copepod reproduction were detected and further ingestion of the cyanobacterium was measured in A. bifilosa using qPCR in the controlled laboratory. The intake of N.
spumigena was positively related to the copepod oxidative status, egg production and their viability, and development of the offspring. To further
evaluate integrated effects of nutritional conditions during summer cyanobacteria blooms on the offspring development, we conducted a follow-up
13
analysis of long-term data on growth conditions for copepod nauplii. This
was done by relating nauplial growth status assessed as their RNA:DNA
ratio to the environmental variables, i.e., temperature, climatic indices, and
phytoplankton, with particular focus on filamentous cyanobacteria. In both
copepod species, this analysis identified cyanobacteria as a significant positive predictor for the nauplial growth status. Our results implicate that in
summer conditions, the toxin producing cyanobacteria N. spumigena and
Anabaena spp. are a valuable food for calanoid copepods and that the diazotrophic cyanobacteria may create a favorable growth environment for nauplii. The favorable growth conditions for nauplii observed during NFC
blooms were hypothesized to be mediated by picoplankton fueled by nitrogen exudates during and after the bloom and in Paper II we tested this hypothesis.
4.2. Picoplankton: a non-existing food source for metazooplankton?
Our current knowledge on the microbial component of zooplankton diet is
limited, and it is generally assumed that bacteria-sized prey (0.2–2.0 µm;
Fig. 1) is not consumed by most metazooplankton grazers in the marine food
webs, because of size constraints on feeding mechanisms (Berggreen et al.
1988, Hansen et al. 1994). However, there are observations that picoautotrophs, including picocyanobacteria, are ingested in situ by mesozooplankton, although the relative importance of this feeding for the grazers remains
unclear (Pace et al. 1990, Wilson and Steinberg 2010). In particular, during
bloom of filamentous algae, poor quality prey frequently dominate phytoplankton (Richardson 1997). Under such conditions, the ability to feed on
microbial components, including picoplankton, would be an advantage for
grazers (Buskey et al. 2003). Among metazooplankters, non-selective feeders (e.g. appendicularians, cladocerans and bivalves) are known to feed on
picoplankton (Weisse 1993, Lipej et al. 1997, Callieri et al. 2012). By contrast, copepods are selective feeders (DeMott 1988) and usually graze larger
food items than cladocerans and appendicularians (Sommer and Stibor
2002). It is also questionable whether this prey is efficiently digested and
assimilated and thus contributes towards production (Azam et al. 1983, Callieri and Stockner 2002).
The positive effects of NFC blooms on egg production and nauplial growth
reported in Paper I raised questions about possible mechanisms of these
effects. As the most plausible mechanism, we suggested that these grazers
may benefit from elevated quantity and quality of picoplankton, which is
stimulated by nutrient exudates from NFC. Indeed, Baltic Sea Aphanizomenon sp. and N. spumigena directly release on average 20–35% of their
fixed N as NH4+, which is then used by other primary producers, e.g., pico14
cyanobacteria, as shown by various approaches including 13C and 15N tracers
(Ohlendieck et al. 2000, Stal et al. 2003, Ploug et al. 2011). However, this
explanation relies on the assumption of high grazing capacity of the nauplii
towards picoplankton.
To evaluate this assumption and substantiate the hypothesis, grazing capacity of Baltic metazooplankton, including juvenile copepod stages, on picocyanobacteria was addressed in Paper II. We used qPCR targeting ITS-1 sequence of the chrococcoid picocyanobacteria Synechococcus to quantify
occurrence of its DNA in the digestive system of zooplankton (rotifers, cladocerans and copepods of different developmental stages) collected during
summer in a coastal area of the Baltic proper. We also related Synechococcus consumption to phytoplankton and NFC stocks during the season. Subsequently, a confirmatory experiment was conducted to:
(1) test whether adults and nauplii of the copepod Acartia tonsa consume picocyanobacterium Synechococcus in the presence of an
alternative food (cryptophyte alga Rhodomonas salina) and in the
absence of ciliates and nanoflagellates,
(2) determine non-consumptive contribution of picocyanobacteria to
zooplankton samples, due to adherence to body surfaces and other sources of contamination and
(3) quantify carbon uptake in the copepods from 13C-labeled
picocyanobacterium S. bacillaris.
All field-collected zooplankton were found to consume Synechococcus spp.
in substantial quantities, indicating that picocyanobacteria are actively utilized by virtually all metazooplankton groups. Moreover, the gut content in
copepods was positively related to the picocyanobacteria abundance and
negatively to the total phytoplankton abundance in the water column at the
time of sampling, indicating opportunistic feeding. It is thus possible that at
low concentrations of preferred prey species, copepods may switch to more
abundant sub-optimal prey (Landry 1981). The experiment with laboratory
reared A. tonsa (older copepodites and nauplii) was fed a mixture of picocyanobacterium S. bacillaris and microalgae R. salina confirmed that copepods
ingested S. bacillaris, even when the alternative food was plentiful, but also
that contamination with the picocyanobacteria contributed ~20% to the total
picocyanobacteria amount in zooplankters. Another important finding is that
picocyanobacteria were not only ingested but also assimilated by the copepods as shown by feeding experiments with 13C-labelled S. bacillaris.
15
4.3. Effects of cyanobacteria on copepod growth
Zooplankton grazers appear to be highly selective in their ability to utilize
cyanobacteria (Haney 1987). However, this selectivity can be affected by
production of toxins and bioactive substances by cyanobacteria as well as by
presence and abundance of alternative prey and physical factors (e.g., temperature and stability of the water column, which influence vertical migrations, etc.). Generally, cyanobacteria have been implicated in conveying
little or no growth benefits for grazers or even causing adverse responses,
such impairment of survival, growth and reproduction of grazers (Wiegand
and Pflugmacher 2005) and oxidative stress (Smith et al. 2008, Amado and
Monserrat 2010). At the same time, cyanobacteria are a rich source of N and
P (Walve and Larsson 2007), essential amino acids (Ahlgren et al. 1992) as
well as some vitamins (Prasanna et al. 2010) and antioxidants (Pandey and
Pandey 2009). When toxic N. spumigena was used as single food source in
short-term laboratory studies, negative effects on zooplankton survival and
reproduction have been reported (Engström et al. 2000, Engström et al.
2001). However, adding a second algal species of sufficient nutritional quality to a diet of cyanobacteria alone has been shown to reduce the negative
effect of cyanobacteria (Reinikainen et al. 1994).
In the Baltic Sea, zooplankton and NFC abundance increase in concert during summer, implying multiple interactions between zooplankton and cyanobacteria. Diazotrophic N from NFC can reach consumers via direct grazing on NFC and through non-diazotrophic phytoplankton fueled by diazotrophic N (Woodland et al. 2013). It is unclear, via which pathway and when
dominates the transfer of diazotrophic N to higher trophic level in the Baltic
Sea. In Paper III, we hypothesized that direct grazing contributes substantially to the incorporation of diazotrophic N by zooplankton and conveys
positive fitness effects. We used field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic proper
and employed combination of approaches to,
(1) quantify amount of Nodularia spumigena and Synechococcus in
zooplankton guts by qPCR,
(2) relate the gut content measurements to changes in δ15N signatures
of zooplankton determined by SIA,
(3) determine the consequences of direct grazing by analyzing zooplankton reproductive and growth indices and
(4) analyze C:N ratio in zooplankton in relation to N content in POM
during cyanobacterial bloom.
16
In the field survey, we found that N. spumigena and Synechococcus spp.
were grazed by all representative zooplankton species, with a significantly
higher size-specific ingestion of Synechococcus spp. in smaller zooplankters
(in line with Paper II findings). The δ15N signatures in zooplankton decreased with both NFC biomass and amount of N. spumigena ingested by the
zooplankters, indicating that cyanobacterium is not only ingested but also
efficiently assimilated. Moreover, growth response (measured as RNA:DNA
ratio) to N. spumigena intake differed between the copepod species, with
negative relationship in Acartia spp. and positive in E. affinis. In both species nauplial growth was positively related to their feeding on Synechococcus spp. This is in line with earlier observed negative effects of feeding on
Nodularia at bloom concentrations on egg production in Acartia (Vehmaa et
al. 2013; Paper I). Picocyanobacteria were positively correlated with biomass of Aphanizomenon spp. and Dolichospermum spp., suggesting that
grazing on picocyanobacteria may indirectly reflect trophic relationships
with these NFC. On the other hand, feeding on picocyanobacteria was negatively correlated with growth response in both Acartia spp. and E. affinis
suggesting nutritional inadequacy or poor assimilation efficiency towards
this prey. The C:N ratio (a proxy for lipid reserves) in zooplankton was significantly positively related to the increasing nitrogen content of POM (<10
µm) and decreasing POM δ15N during the bloom. These findings support the
evidence that cyanobacteria are readily consumed by Baltic Sea zooplankters, including copepods with differential effects on their growth and
development. In a nitrogen-limited environment, ready access to a high N
source that is not generally exploitable by other grazers may convey a selective advantage.
4.4. Diazotrophic cyanobacterial blooms support Baltic productivity
Studies showing that filamentous toxin-producing cyanobacteria are eaten by
many grazers, suspension-feeders and deposit feeders, often with beneficial
effects on their growth and reproduction (Meyer-Harms et al. 1999, Karlson
et al. 2014, Lesutiene et al. 2014) suggest that NFC may support Baltic
productivity. In the Baltic proper, the input of new combined N through N2fixation was estimated to be 20–45% of the yearly average new production
(Gustafsson et al. 2013). The transfer pathways and turnover rates in foodwebs are highly variable and make it hard to quantitatively trace the flow of
combined N from primary producers to top consumers. Studies tracing
source and fate of cyanobacteria-derived N through primary and secondary
trophic levels in the Baltic Sea followed the depleted 15N-signal in many
invertebrates and fish during cyanobacterial blooms (Nordström et al. 2009,
Karlson et al. 2014, Lesutiene et al. 2014), indicating an uptake of diazotrophic N in the food web. In Paper IV, we synthesized recent experimental
17
and field studies addressing utilization of cyanobacterial production in pelagic and benthic food webs that provide empirical evidence that cyanobacterial
nitrogen is efficiently assimilated and transferred in Baltic food webs.
Multiple approaches like SIA, cyanotoxins, pigments, fatty acids (FAs) and
DNA (molecular diet analysis) as trophic markers are convincingly showing
that many pelagic and benthic consumers consume and assimilate diazotrophic cyanobacteria. qPCR analysis has made it feasible to identify and
quantify Baltic cyanobacteria in the gut of consumers as small as nauplii and
rotifers (Papers I, II and III), thus providing in situ data on direct cyanobacteria grazing by zooplankton. Combining molecular diet analysis with
SIA, a decrease in δ15N signal was observed in planktonic grazers when
amount of cyanobacteria in their stomachs increases implicating direct grazing as an important pathway (Papers III and IV). The overall conclusion of
Paper IV is that by utilizing production of NFCs, pelagic and benthic grazers would enhance secondary productivity of the system and food availability for larval, planktivorous and benthic fish. This information is an essential
step towards guiding nutrient management to minimise noxious blooms
without overly reducing secondary production, and ultimately most probably
fish production in the Baltic Sea.
18
5. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The qPCR based detection and quantification of organisms in environmental
samples is particularly useful for direct diet analysis applicable in field and
laboratory studies on zooplankton. The molecular diet analysis provides
direct evidence that mesozooplankton are actively feeding on both filamentous (Nodularia spumigena; Papers I and III) and picocyanobacteria (Synechococcus spp.; Papers II and III). The observed feeding by mesozooplankton on cyanobacteria of different size has important implications for
our understanding of the marine planktonic food webs. The direct pathway
of carbon transfer from picoautotrophs, bypassing multiple trophic transfers
in the microbial loop, implies higher transfer efficiency from primary producers to primary consumers, whereas direct grazing on NFC implies a certain grazing control over summer cyanobacteria blooms and transfer of biomass produced by these blooms to secondary producers. The observed positive effects of Nodularia consumption by the copepods on their recruitment
suggest that NFC contribute directly to zooplankton growth, particularly
during the periods of low availability of preferred phytoplankton prey (Paper I). Similarly, increased consumption of picocyanobacteria with decreasing stocks of larger phytoplankton indicates opportunistic feeding of copepods and certain reliance on picoplankton food.
There were both positive and negative effects of Baltic filamentous cyanobacteria on copepod physiological responses. The toxin-producing bloom
forming cyanobacteria, N. spumigena and Anabaena spp., were grazed upon
and had positive effects on copepod recruitment and growth. Moreover, NFC
were identified as significant positive growth predictors for copepod nauplii.
Whereas viable egg production in the field populations of Acartia was positively related to N. spumigena abundance and no effect was observed in the
laboratory experiment. These differential responses suggest that variations in
the feeding environment may be crucial for cyanobacteria-copepod interactions.
Using field observations on zooplankton and phytoplankton development
during growth season in the northern Baltic proper, we found that cyanobacterial N is assimilated and transferred to zooplankton via both direct grazing
on NFC and indirectly through grazing on small-sized picocyanobacteria that
take up nitrogen exudates (Paper III). The positive relationships between
POM δ15N, C:N ratio and NFC biomass indicate that diazotrophic N fuels
phytoplankton and bacterioplankton communities during summer and improves zooplankton food quality by decreasing POM C:N ratio. In addition,
a positive growth response to grazing on NFC was observed in adult copepod Eurytemora affinis but not in Acartia spp., whereas nauplial growth in
both species was positively related to their feeding on picocyanobacteria.
19
These findings provide strong empirical evidence that both filamentous and
picocyanobacteria are readily ingested and assimilated by zooplankton, with
differential effects on their growth, recruitment and, ultimately, production.
A more general perspective is needed to quantify flows of diazotrophic nitrogen in the ecosystem, including assimilation efficiencies in metazooplankton feeding on filamentous cyanobacteria and picoautotrophs. Should the
energy transfer from NFC and microbial producers to metazooplankton be
efficient, the cyanobacteria blooms may present an important source to secondary and subsequently tertiary production in the Baltic Sea. Our results
warrant a revision of current pelagic food web models linking phytoplankton
to secondary production in the Baltic Sea as well as other systems where
picoplankton contributes substantially to primary production. Further, investigating the associated ecological and evolutionary mechanisms behind cyanobacteria-copepod interactions and adaptive capacities of zooplankton species to noxious blooms would enhance our understanding of ecosystem functioning and associated biogeochemical fluxes in changing environmental
conditions. Studies on the long-term dynamics of cyanobacteria and grazers
in the Baltic Sea in relation to the environmental factors are also highly relevant for improving our predictive capacity through retrospective analysis
using various statistical methods. Furthermore, more controlled experiments
with semi-natural communities, combined research methods and frequent
sampling schedules are needed to pinpoint the specific interactions and responses that contribute to the observed long-term dynamics and evolutionary
adaptations.
20
6. ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to thank those who have
contributed to this thesis and supported me in one way or the other during
this amazing journey.
First of all, I am extremely grateful to my supervisor, Prof. Elena
Gorokhova. Her intellectual guidance, motivation and profound knowledge
of the field encouraged me to carry on through these years. The useful
discussions and brainstorming sessions that we have had helped me at
various stages of my research. I truly appreciate all her time, contributions
and encouragement that led me to work on diverse exciting projects. Elena, I
thank you wholeheartedly for accepting me as a PhD student and for the
whole journey.
I extend my thanks to my former co-supervisor Dr. Anna Edlund for the
insightful discussions on Paper II. Thank you co-supervisor Dr. Jakob
Walve, for all valuable comments. I am thankful to all my co-authors and
Dr. Jonna Engström-Öst for a great collaboration. I am grateful to Dr.
Helena Höglander, for kindly helping me with the microscope related queries and sharing pictures of cyanobacteria and copepods. Thank you Prof.
Emeritus Ragnar Elmgren, for keeping interesting articles in my mail-box
throughout these years.
Thank you Jennie Svéden for being an amazing friend over these years, sharing the office with you has been great fun with all the discussions, both scientific as well as non-scientific. Special thanks for your help with Swedish–
English translations! Thanks to Jon Duberg, it has been a pleasure working
with you. All other friends and colleagues at the department are thanked for
being helpful and creating a wonderful atmosphere at the work place.
Thanks also to late Leif Lundgren, Stefan Svensson and Bengt Abrahamsson
for collecting samples.
To all my teachers and friends back home in India, thank you for sharing
your knowledge and encouragement in many ways. My sincere thanks goes
to Dr. Sadhana Rayalu for nurturing my enthusiasm in science. Apologies
for not listing all the names here, but you are always in my mind. Thank you
for kindly sharing your photos for cover picture, Elena G for zooplankton
pictures and Tomislav Lukman for DNA picture. I greatly appreciate the
help from Navin Varma, for putting up the cover picture layout.
I would like to extend my heartfelt gratitude towards my family: My grandmother, parents, and family-in-law for their encouragement in all my pursuits. My parents, for being great source of inspiration and I dedicate this
21
thesis to them. Pankaj, Mukesh, Bhavesh and Bhavik, for laughs and affection. Even though you all are far away from me, I always felt your love,
guidance, support and blessings. I greatly appreciate the support from my
loving parents-in-law, especially during last year. Finally, but by no means
least, my heartfelt thanks towards my beloved husband Hitesh, for his unwavering love, patience and continual support of my academic endeavours. Our
lovely daughter, Gitarika, who is the sunshine of my life and has given me
the extra strength and motivation to get things going.
Few lines in Hindi,
मेरे मम्मी-पापा को विशेष धन्यिाद, उनके प्रोत्साहन और अंतहीन प्यार के लिए, जो
उन्होंने मुझे िषों से ददया है । मैं इस सफिता का श्रेय अपने मम्मी-पापा को दे ती हूँ।
अम्मा और सभी बड़ो का आर्शीवाद हमेर्शा बना रहे ।
Financial support was received from The Swedish Research Council for
Environment, Agricultural Sciences and Spatial Planning (FORMAS) and
Stockholm University’s strategic marine environmental research program
“Baltic Ecosystem Adaptive Management”.
22
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