Nutritional aspects in the invasive freshwater bivalve Corbicula fluminea : The role of essential lipids

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Nutritional aspects in the invasive freshwater bivalve Corbicula fluminea:

The role of essential lipids

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von

Basen, Timo

Tag der mündlichen Prüfung: 27.07.2012

1. Referent: Prof. Dr. Karl-Otto Rothhaupt

2. Referent: Prof. Dr. Alexander Wacker

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Ernst Jandl

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Chapter 1 General introduction

Autecology of Corbicula fluminea

The Asian clam Corbicula fluminea (OF Müller 1774) is a hermaphroditic self fertilizing clam with a life span of 1 - 5 years (McMahon 2002; Sousa et al. 2008). The clams start to reproduce at a shell size of about 10 mm (i.e. at the age of 5 - 9 months); between one and three reproduction cycles are possible depending to ecosystem conditions. Adult Corbicula have a high fecundity and conduct brood hatchery (Kraemer and Galloway 1986; Araujo et al. 1993), whereat larvae are kept in inner demibranches until they reach a size of approximately 250 µm. The larvae (Fig. 1c) are then released into the water column and settle to the ground within 24 h, i.e. the time span for drift distribution is limited. A temperature tolerance of > 2 and < 36 °C has been reported (Britton and Morton 1979;

Karatayev et al. 2005), but longer periods with temperatures about 3 °C (Werner and Rothhaupt 2008) and times with low food supply and high temperatures during summer months have been shown to cause high mortality rates in C. fluminea (Weitere et al. 2009;

Vohmann et al. 2010). At a temperature of 10 - 11 °C growth and reproduction are possible (Karatayev et al. 2005). Clams prefer habitats with soft sediments (small gravel, sand) which are well oxygenated and contain high proportions of organic matter (Hakenkamp and Palmer 1999; Vaughn and Hakenkamp 2001). Juvenile and adult clams are capable of deposition feeding, the so called ‘pedal feeding’ (Reid et al. 1992), which is considered as the primary mechanism of larval nutrition until gill structures and in- and exhalant siphons are developed properly. However, although adult Corbicula are still able to take up food via its muscular foot, filter feeding clearly is the main process of food uptake in the Asian clam.

Fig. 1: Different stadia of Corbicula fluminea. Adult clams with closed shells (a), with expelled siphons and muscular foot (b) and D-shaped larvae (size approximately 300 µm; c).

a b c

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The clams circulate water for respiration and feeding, and remove particles from the water, which are either consumed or bound as pseudofaeces and expelled. Corbicula fluminea can effectively remove detritus, bacteria and algae from the water column but is regarded as a non-selective suspension feeder (Lauritsen 1986; Way et al. 1990; Boltovskoy et al. 1995).

Preferred size range for ingestion is known to be between 1 μm and 20 - 25 μm (Way et al.

1990; McMahon and Bogan 2001), with reported maximal values of up to 170 μm (Boltovskoy et al. 1995).

Ecological impacts of C. fluminea

Bivalves can dramatically alter the structure of benthic communities. This was shown for the zebra mussel Dreissena polymorpha in Lake Constance in the 1960s (Mörtl and Rothhaupt 2003) and in other freshwater ecosystems in Europe and North and South America (Stanczykowska 1977; MacIsaac et al. 2002). One contributing factor is the physical alteration of habitat structure, the so-called ecosystem engineering (Jones et al. 1994).

Bivalve shells can provide shelter and substrate for other benthic species (Strayer et al. 1999;

Crooks 2002; Sousa et al. 2008). Burrowing bivalves, like C. fluminea, can impact benthic processes such as nutrient and organic matter cycling in sediments (Hakenkamp and Palmer 1999; Vaughn and Hakenkamp 2001). Additionally, filter feeding bivalves are often considered as important benthic-pelagic couplers, which remove large amounts of seston (algae, bacteria, particulate organic carbon) from the water column and transfer these resources to the benthos as biodeposition material (faeces and pseudofaeces), thereby stimulating benthic productivity (Hakenkamp et al. 2001; Gergs et al. 2009). Filtration processes mediated by bivalves can severely disturb the recruitment of other bivalves species (Hakenkamp and Palmer 1999), when larval swimming stadia are ingested (Strayer et al. 1999). Additionally, a high filtration rate can regulate pelagic nutrient cycling (Cohen et al.

1984; Cahoon and Owen 1996; Hwang et al. 2011), reduce eutrophication processes (Phelps 1994; McMahon 2002) and increase water clarity and therefore promote submerged vegetation (Phelps 1994). Moreover, the excretion of inorganic nutrients to the water column by filter feeding may play an important role in stimulating primary production (Yamamuro and Koike 1993; Vaughn et al. 2008). Filtration, nutrient excretion and benthic- pelagic coupling are regarded as the main water column processes completed by C. fluminea (Lauritsen and Mozley 1989; Vaughn and Hakenkamp 2001). A high filtration rate favours Corbicula in competition with other bivalves (Sousa et al. 2005), which has been shown for German water ways, where Corbicula massively spread and quickly replaced D. polymorpha as dominant mollusc (Tittizer et al. 2000; Bachmann et al. 2001)

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Fig. 2 Map of Lake Constance (after IGKB 2010). Numbers indicate the site of first occurrence of Corbicula fluminea in the lake in 2003 (1) and the maximal distribution on the northern shore up to Immenstaad (2) and on the southern shore up to Altnau (4) in 2010. An isolated discovery was detected in the “Überlinger See” in 2008 (3). In 2011 first observations of C. fluminea were recorded in the “Seerhein” near the city of Konstanz (5).

Invasion history of C. fluminea

Since its introduction into many aquatic freshwater systems all over the world during the last decades, C. fluminea has undergone a remarkable range expansion to become a ubiquitous benthic invertebrate in freshwater ecosystems. Originating from Southeast Asia, C. fluminea was introduced to North America (McMahon 1982) and South America (Darrigran 2002) in the early 20^thcentury. In the 1980s Corbicula spp. invaded Europe, presumably via ballast water used in ships originating from North America (Mouthon 1981), and this event was followed by a rapid expansion in European inland waters (Den Hartog et al. 1992; Araujo et al. 1993). In 1988 it was also first detected in the River Rhine delta (Bij de Vaate and Greijdanus-Klaas 1990). Within years it inhabited the whole navigable River Rhine up to the Swiss border (Turner et al. 1998; Tittizer et al. 2000). The first individuals of C. fluminea in Lake Constance were discovered at the Rohrspitz (Austria) in 2003 (Fig. 2; position1; Werner and Mörtl 2004). In the following years the clams spread within the eastern part of the lake.

Further occurrence of C. fluminea was discovered at the southern shore (Switzerland) and at the northern shore (Austria, Germany) of Lake Constance. In 2008, C. fluminea spread along the northern shore up to Friedrichshafen (Fig. 2, position2), an isolated appearance in the western part of the lake close to Konstanz-Egg (3) was also discovered. The maximum

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western distribution in 2010 reached the axis Altnau (4) – Friedrichshafen (2). In 2011, first observations of C. fluminea were reported near the city of Konstanz (“Seerhein”, 5). A further establishment of C. fluminea in the lower Lake Constance (Untersee) is merely a matter of time.

Lake Constance is an oligotrophic pre-alpine lake, situated in Middle Europe, with Germany, Switzerland and Austria as riparian states. With a maximum depth of 253 m, a surface of 473 km², and ~50 km³ water volume, Lake Constance is one of the largest and deepest lakes in Middle Europe. The lake consists of two basins, the larger and deeper Upper Lake and the smaller and shallower Lower Lake Constance. The major contributing river is the Alpine Rhine, located in the eastern part, in Austria. First specimens of C. fluminea in Lake Constance were found in a shallow, sandy bay between the old and new Alpine Rhine river bed (Fig. 2, position1; Fig. 3; Werner and Mörtl 2004).

The C. fluminea population in Lake Constance is characterized by slow growth, a reduced maximum shell size, and only one reproductive period per year (Werner and Rothhaupt 2008). At the Rohrspitz (Austria), the site of maximum density, up to 90% of total biomass of littoral community was represented by C. fluminea (Werner and Rothhaupt 2007).

Fig. 3: Impressions from the sampling site of Corbicula fluminea at the Rohrspitz (Austria; a), collected clams with gravel and debris (b) and underwater pictures of typical clam habitats (c-e; © 2012 John Hesselschwerdt - RHEOS).

a b

c d e

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Many invasions in Lake Constance took place in the last decades, with successful invasions of gastropods (Viviparus ater), crayfish (Orconectes limosus), amphipods (Dikerogammarus villosus), mysids (Limnomysis benedeni ) and bivalves like D. polymorpha and C. fluminea (for details see Hanselmann 2011). Most non-native species do not successfully establish or have only little impact on communities.However, the introduction of new species to ecosystems is potentially associated with a loss of diversity and ecosystem stability (Sala et al. 2000;

Chandra and Gerhardt 2008; Strayer 2010). In the last decades, the spread of invasive species in new freshwater ecosystems rapidly increased (Richardson and Pysek 2008).

Especially in lakes, invaders were identified as the main cause of extinction of native species (Lodge 2001; Strayer 2010). Invasive species can disturb food web processes, introduce diseases and parasites and alter the natural species composition (Tittizer et al. 2000; Ellis et al. 2011; Poulin et al. 2011). Hence, it is important to understand the factors which are responsible for the successful geographic spread of these invasive species in order to predict possible expansions and to assess ecosystem consequences.

How to be an invasive clam?

One main factor limiting expansion of C. fluminea is water temperature. It has been suggested that anthropogenic increase of minimum temperature in winter, caused by power plants (cooling water outflow), can favour the successful establishment in German water ways (Schöll 2000). Additionally, climate change is known to favour species adapted to warmer temperatures or prone to extreme cold temperatures (Mooij et al. 2005). For the pelagic food chain severe effects are postulated to show up with global warming, e.g.

increase in frequency of cyanobacterial bloom formation (Paerl and Huisman 2008; Wagner and Adrian 2009). Climate scenarios with rising temperatures and increased atmospheric CO2 supplies are expected to favour cyanobacterial dominance, because cyanobacteria potentially have a competitive advantage over other phytoplankton groups in coping with the expected increase in abiotic challenges (Jöhnk et al. 2008; Paerl and Huisman 2009;

Wagner and Adrian 2009; Paerl et al. 2011). Increased periods of thermal stratification have been suggested to shift phytoplankton assemblages towards a higher proportion of cyanobacteria capable of N2-fixation, such as Aphanizomenon or Anabaena, which may also affect predator-prey interactions in aquatic food webs (Wagner and Adrian 2011).

Cyanobacterial blooms can be associated with hazards to human health and livestock and reduced quality of water bodies (Carmichael 1992; Codd 1995; Paerl et al. 2011). Therefore, it is important to investigate the consequences of cyanobacterial mass development for the aquatic food web.

The elementary process in food webs is the transfer of energy from one trophic level to the next. Most important for the carbon flow in aquatic food webs is the plant-herbivore interface, at which the transfer efficiency is highly variable and affected by many factors.

Structure and morphology of planktonic cells can affect food quality (DeMott et al. 2001;

Van Donk et al. 2011). Digestion efficiency is also a factor determining food quality, e.g.

when cells are imbedded in gelatinous layers (Van Donk and Hessen 1993; Van Donk et al.

2011). However, not only morphology but also the compounds within the cells have to be

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considered in the context of food quality. Secondary metabolites, such as toxins, affect primary consumers when feeding on toxic strains of phytoplankton (Lampert 1987;

Carmichael 1992). Various taxa of eukaryotic algae and especially cyanobacteria produce toxins that greatly reduce their nutritional value for herbivores. Selective feeding bivalves are known to promote blooms of toxin producing cyanobacteria and additionally increase the benthic pelagic coupling, due to high rates of pseudofaeces production and low digestion rates (Vanderploeg et al. 2001; Pires et al. 2005; Bontes et al. 2007). Besides toxins, protease inhibitors may also reduce the digestibility of ingested food sources and thus may reduce food quality for filter feeding cladocerans (Schwarzenberger et al. 2010).

Furthermore, the lack or low availability of essential nutrients can constrain the quality of food. Among mineral nutrients, nitrogen and phosphorus are of particular importance in aquatic ecosystems (Elser et al. 2000; Sterner et al. 2008). In marine ecosystems, nitrogen is known as the main limiting parameters for energy transfer between trophic levels (Tyrell 1999), whereas in freshwater systems phosphorus is considered to be the most important limiting nutrient (Elser et al. 2000). Somatic growth especially depends on the supply with phosphorus, because growth is linked to the protein synthesis rate and thus to the concentration of phosphorus-rich ribosomal RNA (Elser et al. 2003a). In autotrophs the C:N:P ratio is known to be highly variable, whereas consumers tend to maintain homeostasis, i.e.

relatively constant body C:N:P ratios (Hessen and Lyche 1991; Frost et al. 2002; Elser et al.

2003b). Thus, insufficient availability of N and P for herbivores, i.e. high C:N and C:P ratios in primary producers, can lead to limitation of herbivore growth (Urabe and Watanabe 1992;

Sterner 1993). However, most of the studies in freshwater ecosystems focused on interactions between planktonic organisms. Within the last years, stoichiometric demands in the benthic food web has come into focus (Frost et al. 2002; Frost et al. 2003; Cross et al.

2005; Fink et al. 2006; Gergs and Rothhaupt 2008). However, the importance of N and P limitation in benthic food webs is still barely understood, especially for bivalves. In addition, new challenges in food webs mediated by climate change or introduction of non-native species disturb established patterns and therefore might influence micronutrient effects.

Reports on food quality effects on freshwater bivalves, apart from those mediated by mineral nutrient stoichiometry, are scarce. In a previous attempt, Foe and Knight (1986) examined the growth of C. fluminea on six genera of green algae provided in various combinations, and recorded positive tissue growth on different mixtures of Ankistrodesmus, Chlamydomonas, Chlorella, and Scenedesmus, but not for unialgal food sources or mixtures containing Pedinomonas or Selenastrum. However, requirements of Corbicula for single nutrients have not been further characterized. Corbicula, like filter feeding daphnids, are known to be non-selective filter feeders and thus are unable to select food particles with regard to their quality. Therefore, they might respond particularly sensitively to the predominance of nutritionally inadequate food sources (e.g. lipid or micronutrient depleted algae). Research on marine bivalves used for aquaculture revealed that growth and reproduction of bivalves is significantly affected by dietary lipids (e.g. Delaunay et al. 1993;

Soudant et al. 1996a; Knauer et al. 1999; Ben Kheder et al. 2010). For the freshwater bivalve

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D. polymorpha, Wacker and Von Elert (2002; 2003; 2004) reported evidence that growth, survival and juvenile recruitment are affected by the dietary essential fatty acid supply, especially the supply with long chain polyunsaturated fatty acids (PUFAs) like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). PUFAs play an important role in the physiology of animals (Cook 1996). They are components of cell membranes and precursors of bioactive molecules, such as eicosanoids (Stanley-Samuelson 1994), and cannot be synthesized by animals de novo, and thus are essential for their nutrition.

Sterols are another class of lipids that are required for a multitude of physiological processes, e.g. as indispensable components of cell membranes, as precursors for vitamin D and steroid hormones and for regulation of signal transduction (for more detail see Martin- Creuzburg and Von Elert 2009). The ability to synthesize sterols de novo is related to eukaryotic cells, whereas in prokaryotes like cyanobacteria, sterols are usually absent (Volkman 2003; Summons et al. 2006). Cholesterol is the predominant sterol found in animals (Goad 1981). In aquatic ecosystems the role of sterols for food quality was first investigated in filter feeding zooplankton. Arthropods are not capable of synthesising sterols de novo and thus require a dietary source of sterols for growth and reproduction (Martin- Creuzburg and Von Elert 2009). The absence of dietary sterols impairs the fitness of filter feeding zooplankton, as has been shown by sterol supplementation of sterol deficient food sources (Hassett 2004; Martin-Creuzburg and Von Elert 2004). So far, sterol demands in benthic invertebrates have not been investigated. Experiments with marine bivalves, mostly with species used in aquaculture, suggest that the ability to synthesize sterols de novo is generally low or absent among bivalve species, which implies that a dietary source of sterols is necessary for growth (Voogt 1975; Soudant et al. 2000; Park et al. 2002). Data on sterol requirements of freshwater bivalves have not been collected yet.

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Fig. 4 Impressions of experimental setups in field and laboratory experiments: (a) plastic boxes used for the determination of clam growth in Lake Constance (b); different algae (d) and clam cultivation vessels (c, e, f) used in laboratory experiments.

Aims and objectives

To elucidate the energy flow in freshwater ecosystems it is important to understand the factors affecting the efficiency of energy transfer across trophic levels. My work was focused on food quality effects on the pelagic-benthic coupling, with key aspect on the nutrition of the benthic freshwater clam C. fluminea.

The thesis starts with two laboratory studies about the role of essential biochemicals in clam nutrition. At first, nutritional requirements of C. fluminea were investigated in a standardized experimental setup, where food and clam tissue parameters (fatty acids, sterols, elemental stoichiometry) were estimated and related to somatic growth rates of individual clams (chapter 2). Phytoplankton species were used which differed in their morphology and in their biochemical composition to assess the significance of different food quality constraints for the growth of clams.

a b

c

d e f

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Based on these results, the role of sterols for growth of C. fluminea became a focal point of this study. In a second experimental approach clams were fed with different sterol containing eukaryotic algae and sterol-free cyanobacteria diets (chapter 3). A supplementation method was developed to enrich cyanobacteria with sterols to be able to investigate whether growth conditions for C. fluminea are constrained by a deficiency in dietary sterols.

To assess the significance of environmental factors on growth and survival of C. fluminea in Lake Constance, an experimental field study was performed in Lake Constance during the year 2010 (chapter 4). I hypothesized that seasonal variations in water temperature, phytoplankton succession and seston nutrient composition influence clam growth.

Additionally, concomitant laboratory experiments were conducted to study the influence of temperature on clam growth rates and to compare the physiological states of C. fluminea collected from the field in different phases during the season.

Corbicula fluminea is an important pelagic-benthic coupler and thus might be able to transfer not only energy but also essential nutrients to detritivorous benthic invertebrates when they feed on clam-generated biodeposition material. Therefore, I explored the mechanisms responsible for growth and survival of a benthic invertebrate, i.e. Gammarus roeselii, which I fed with different sestonic phytoplankton species and clam generated biodeposition material. The benthic-pelagic coupling mediated by C. fluminea was investigated in an experimental food chain in laboratory experiments (chapter 5), where the role of bioavailability, micronutrients and biochemicals in pelagic autotrophs and clam generated biodeposition materials for gammarid growth and survival was examined.

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Chapter 2 Role of essential lipids in determining food quality for the invasive freshwater clam Corbicula fluminea

Timo Basen, Dominik Martin-Creuzburg and Karl-Otto Rothhaupt

Freshwater Science (formerly Journal of the North American Benthological Society) doi: 10.1899/10-087.1

Received: 23 June 2010 Accepted: 16 March 2011

Abstract

The invasive clam Corbicula fluminea has become a widespread benthic invertebrate in many freshwater ecosystems throughout Europe and North and South America. Invasive bivalves can dramatically alter the structure of native benthic communities, so understanding the factors responsible for successful invasion is important. We investigated C. fluminea nutritional requirements for essential lipids in a standardized growth experiment. Juvenile clams were fed different cyanobacteria (Aphanizomenon flos-aquae, Anabaena variabilis, Synechoccocus elongatus) or eukaryotic algae (Scenedesmus obliquus, Cryptomonas sp.). Somatic growth rates were then correlated with elemental (C:N and C:P) and biochemical (sterol and fatty acid content) components of the food sources and clam tissue. Somatic growth rates were significantly higher when juveniles were fed eukaryotic algae than when fed cyanobacteria. Linear regression analyses revealed significant positive relationships between somatic growth rates and dietary sterol and polyunsaturated fatty acid content. Somatic growth rates also were highly correlated with the total sterol and α- linolenic acid content of clam tissues. This result suggests that the growth of C. fluminea is partially dependent on the availability of these essential lipids in the diet. Algal nutritional value may influence the successful geographic spread of this highly invasive species because food quality and quantity are changing as a result of global warming.

Key words: Corbicula fluminea, sterol, food quality, cyanobacteria, PUFA, invasive species, essential lipids.

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Introduction

The clam Corbicula fluminea (Bivalvia, Corbiculidae), indigenous to Australia, Asia, and Africa, has been introduced to Europe and North and South America in the last century (Britton and Morton 1979; Araujo et al. 1993). Since its introduction, C. fluminea has undergone a remarkable range expansion to become an ubiquitous benthic invertebrate in freshwater ecosystems. In Germany, C. fluminea has successfully invaded many rivers, streams, and lakes where it often maintains large populations. Corbicula fluminea was discovered in the lower river Rhine in 1988, spread rapidly upstream, and was first recorded in Lake Constance in 2003 (Werner and Mörtl 2004). Invasive bivalves can dramatically alter the structure of native benthic communities as evidenced by the results of invasions of C. fluminea (Aldridge and McMahon 1978; Belanger et al. 1990; Williams et al. 1993) and the zebra mussel Dreissena polymorpha in many freshwater ecosystems in Europe and North and South America (Stanczykowska 1977; MacIsaac et al. 2002; Mörtl and Rothhaupt 2003).

Bivalves like C. fluminea and D. polymorpha are important benthic–pelagic couplers that remove large numbers of particles from the water column and transfer these resources to the substrate as biodeposits (faeces and pseudofaeces), thereby stimulating benthic productivity (Hakenkamp et al. 2001; Gergs et al. 2009). Corbicula fluminea and D.

polymorpha also may play an important role in stimulating primary production by excreting inorganic nutrients to the water column (Yamamuro and Koike 1993; Vaughn et al. 2008).

Hence, it is important to understand the factors responsible for the successful geographic spread of these invasive species.

Invasion patterns are affected by abiotic challenges, such as temperature, turbidity, dissolved O2, or NH3 (McMahon 1979; Cooper et al. 2005; Walther et al. 2009). They also might be affected by food and substrate availability (Foe and Knight 1985; Mouthon 2001;

Schmidlin and Baur 2007) or by availability of essential nutritional components.

Long-chain polyunsaturated fatty acids (PUFAs) are essential dietary compounds that have important physiological functions (Cook 1996). For example, arachidonic acid (ARA) and eicosapentaenoic acid (EPA) are precursors of eicosanoids, which are thought to be relevant to bivalve reproduction and osmoregulation (Stanley-Samuelson 1994). Research on marine bivalves (almost exclusively on species relevant for aquaculture) has shown that a dietary source of certain PUFAs is required for proper growth and development (Delaunay et al.

1993; Soudant et al. 1996b). However, lipid requirements of freshwater bivalves are poorly understood. Recent research showed that certain long-chain PUFAs (e.g., EPA and docosahexaenoic acid [DHA]) are important to D. polymorpha (Vanderploeg et al. 1996;

Wright et al. 1996; Wacker et al. 2002; Wacker and Von Elert 2003; 2004).

Sterols are another class of lipids required for many physiological processes. They are indispensable components of cell membranes and precursors for steroid hormones (Goad 1981; Martin-Creuzburg and Von Elert 2009). Experiments with marine bivalves suggest that the ability to synthesize sterols de novo is generally low or absent among bivalves. Thus, a

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dietary source of sterols is necessary for growth (Soudant et al. 1998; Park et al. 2002).

However, data on sterol requirements of freshwater bivalves are not yet available.

We investigated the nutritional requirements of the invasive filter-feeding freshwater clam C. fluminea for essential lipids in standardized growth experiments. Corbicula fluminea were fed common cyanobacterial and eukaryotic pelagic food sources that differed in their biochemical composition, and their growth rates were related to the availability of essential lipids in their diets and to lipids incorporated in clam tissues.

Methods

Clam collection

Corbicula fluminea individuals were collected in the upper basin of Lake Constance at a sampling site described by Werner and Rothhaupt (2008). The clams were collected by SCUBA divers at water depths of 1.5 to 3 m, depending on lake-level fluctuations. Living individuals were separated from debris, sand, and gravel. They were transferred to flow- through systems with filtered (<30 µm), aerated lake water and precombusted sediment at an ambient temperature of 20°C until the start of growth experiments.

Cultivation of cyanobacteria and algae

Food sources for C. fluminea were cultivated semi continuously in aerated 5-L vessels at a dilution rate of 0.25/d at 20°C with illumination at 100 to120 μmol quanta m^–2 s^–1 and were harvested in the late-exponential-growth phase. The coccoid cyanobacterium Synechococcus elongatus (SAG 89.70; Sammlung für Algenkulturen, Göttingen, Germany), the filamentous cyanobacteria Anabaena variabilis (ATCC 29413; American Type Culture Collection, Manassas, Virginia), and Aphanizomenon flos-aquae (CCAP1401-1; Culture collection of algae and protozoa, Oban, Scotland), and the green alga Scenedesmus obliquus (SAG 276-3a) were grown in Cyano medium (Jüttner et al. 1983). The flagellate Cryptomonas sp. (SAG 26.80) was grown in modified Woods Hole (WC) medium (Guillard 1975) enriched with vitamins and P. These foods were used because they differ in PUFA and sterol content. Food suspensions were prepared by concentrating the cells by centrifugation and resuspension in fresh media. Concentrations of the food suspensions were estimated from photometric light extinction (800 nm) and from C-extinction equations determined prior to the experiments.

Growth experiments

Adult bivalves expend most of their energy in reproduction (gametogenesis) and little in somatic growth (Soudant et al. 1999). Therefore, we used juveniles that were sexually immature to maximize somatic growth rates and to enlarge possible differences in response among food sources (initial fresh mass 70.0 ± 55.1 mg, range between 10.5 - 222.9 mg). The

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28-d experiments were carried out at 20 °C in glass beakers filled with 200 mL of filtered lake water (0.45-μm pore-size membrane filter) and precombusted sediment (550 °C for 5 h) to allow the clams to burrow. Each of the 5 food treatments was replicated in 15 beakers, and 1 randomly chosen clam was transferred to each beaker. Clams were fed daily with 3 mg C/L of the food suspensions during the experiment. Water was exchanged daily to remove fecal pellets, and sediment was exchanged once a week to reduce biofilm formation. No clams died during the experiment.

Somatic growth rates (g) were determined as the increase in dry mass from the beginning of the experiment (W0) to day 28 (Wt) over time (t) with the equation used by Martin–

Creuzburg et al. (2005b):

t W g (lnW^t ln ₀)



.

A subsample of clams (n = 48) was taken at the beginning of the experiment to estimate the individual fresh (FM) and dry mass (DM) after 24 h freeze-drying to establish a fresh-dry- mass regression (DM = 0.599FM). Samples were weighed on an electronic balance (±0.1 μg;

XP2U, Mettler Toledo GmbH, Gießen, Germany). The start-dry mass of clams used in the growth experiment was estimated from their actual fresh mass and the previously determined fresh-dry-mass regression. Growth rates of all specimens were calculated as means (n = 15) for each treatment.

Analyses of food organisms and clam tissues

Aliquots of the food suspensions were filtered onto precombusted glass-fiber filters (Whatman GF/F, 25-mm diameter) and analyzed for particulate organic C (POC) and N with an NCS-2500 analyzer (ThermoQuest, Biberach, Germany). Particulate P was measured in aliquots collected on acid-rinsed polysulfon filters (HT-200; Pall, Dreieich, Germany) and digested with a solution of 10% potassium peroxodisulfate and 1.5% sodium hydroxide for 60 min at 121°C. Soluble reactive P was determined with the molybdate-ascorbic acid method (APHA 1985).

Soft tissues of freeze-dried clams were isolated and weighed before determination of elemental composition. The C and N content of soft tissues (n = 3 per treatment) was measured as described above with an NCS-2500 analyzer. C and N contents were expressed per unit tissue dry mass and were converted to molar C:N ratios. The P in soft tissues (n = 3 for each treatment) was solubilized by mechanical shearing with a mortar and by sonication in ultrapure water. P content was measured as described above. P content was expressed per unit tissue dry mass and was converted to C:P molar ratios.

Lipids in food sources were extracted twice in a mixture of dichloromethane/methanol (2:1, volume/volume [v/v]) from precombusted GF/F filters (Whatman, 25-mm diameter) loaded with ~0.5 mg (for fatty acid analysis) or ~1 mg (for sterol analysis) POC of the algal food

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sources. Soft tissues of freeze-dried clams (n = 2 for each treatment) were separated from their shell, weighed, and placed in dichloromethane/methanol (2:1, v/v). Lipids in soft tissues were solubilized by mechanical shearing with a mortar and by sonication. Half of the resulting tissue solution was used for fatty acid analysis and the other half was used for sterol analysis.

For analysis of sterols, the pooled cell-free extracts were dried under a stream of N2 and saponified with 0.2 mol/L methanolic KOH (70°C, 1 h). Subsequently, sterols were partitioned into iso-hexane:diethyl ether (9:1, v/v), dried under a stream of N2, and resuspended in a volume of 10 to 30 mL iso-hexane. For analysis of fatty acids, the cell-free extracts were dried under a stream of N2 and esterified with 3 mol/L methanolic HCl (60°C, 20 min). Subsequently, fatty acid methyl esters (FAMEs) were partitioned into iso-hexane, dried under a stream of N2, and resuspended in a volume of 25 to 100 mL iso-hexane. Lipids were analyzed by gas chromatography on an HP 6890 gas chromatograph (GC; Agilent Technologies, Böblingen, Germany) equipped with a flame ionization detector and either a DB-225 (J&W Scientific, Cologne, Germany) capillary column to analyze FAMEs or an HP-5 (Agilent) capillary column to analyze sterols. Details of GC configurations are given elsewhere (Martin-Creuzburg et al. 2009; 2010).

Lipids were quantified by comparison to internal standards (C17:0 and C23:0 methyl esters, 5α-cholestane). The detection limit was ~20 ng of sterol/fatty acid. Lipids were identified by their retention times and their mass spectra, which were recorded with a GC/mass spectrometer (GCQ, Thermo Finnigan MAT, Bremen, Germany) equipped with a fused Si capillary column (DB-225MS, J&W Scientific for FAMEs; DB-5MS, Agilent for sterols). Sterols were analyzed in their free form and as their trimethylsilyl derivatives. Mass spectra were recorded between 50 and 600 amu in the electron impact ionization mode, and lipids were identified by comparison with mass spectra of reference substances purchased from Sigma- Aldrich (Munich, Germany) or Steraloids (Newport, RI, USA), mass spectra found in a self- generated spectra library, or in the literature (e.g. Toyama et al. 1952; Belanger et al. 1973;

Goad and Akihisa 1997). The absolute amount of each lipid was quantified per mg POC or soft-tissue C content of clams. Fatty acids are reported using shorthand nomenclature as follows: a:bn-x, where a represents the number of C atoms, b is the number of double bonds, and x is the position of the first double bond counted from the methyl end.

Statistical analyses

All statistical analyses were done with the statistical software package R (R Development Core Team 2006). Differences among growth rates were analyzed with analysis of covariance (ANCOVA) with food treatment as the categorial variable and

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Tab. 1 Mean (±1 SD; n = 3) elemental and biochemical composition of cyanobacteria (Synechococcus elongatus, Anabaena variabilis, Aphanizomenon flos-aquae) and eukaryotic algae (Scenedesmus obliquus, Cryptomonas sp.) used as food sources for Corbicula fluminea. Elemental ratios for C:N and C:P are molar, fatty acids (FA) are reported as µg FA/mg C. Lipid classes are saturated (SAFA), mono- (MUFA), and polyunsaturated fatty acids (PUFA) and fatty acids with double bond at n-3 position, n-6 position, and ratio between those lipid classes (n- 3:n-6). Fatty acids are described as a:b(n-c) where a = number of C atoms, b = number of double bonds, c = position of the first double bond counted from the methyl end. n.d. = below detection limits.

Variable S. eolongatus A. variabilis A. flos-aquae S. obliquus Cryptomonas sp.

C:N 4.4 ± 0.1 4.4 ± 0.1 4.9 ± 0.1 5.8 ± 0.1 6.0 ± 0.1

C:P 53.0 ± 0.3 50.7 ± 1.0 80.2 ± 0.4 40.7 ± 3.0 124.5 ± 3.5 18:2(n-6) n.d. 38.5 ± 1.2 11.1 ± 1.3 46.9 ± 3.1 21.8 ± 1.4 18:3(n-3) n.d. 66.4 ± 2.3 66.2 ± 9.8 134.9 ± 9.5 76.0 ± 5.0

18:4(n-3) n.d. n.d. n.d. 11.8 ± 0.6 36.50 ± 1.7

20:2(n-6) n.d. n.d. n.d. n.d. n.d.

20:4(n-6) n.d. n.d. n.d. n.d. n.d.

20:5(n-3) n.d. n.d. n.d. n.d. 46.9 ± 2.0

22:5(n-3) n.d. n.d. n.d. n.d. n.d.

22:6(n-3) n.d. n.d. n.d. n.d. 5.0 ± 0.1

Total FA content 125.0 ± 11.0 242.5 ± 8.3 157.2 ± 13.9 272.3 ± 22.0 251.2 ± 14.0 SAFA 56.4 ± 8.9 70.6 ± 2.4 55.01 ± 2.50 52.05 ± 3.5 42.4 ± 4.6 MUFA 68.6 ± 2.1 67.1 ± 1.8 24.9 ± 1.6 23.5 ± 3.6 27.5 ± 1.3 PUFA n.d. 104.9 ± 4.4 77.3 ± 11.1 196.7 ± 15.8 181.2 ± 9.8

n-3 n.d. 66.4 ± 3.2 66.2 ± 1.3 146.8 ± 5.7 159.4 ± 1.4

n-6 n.d. 38.5 ± 2.3 11.1 ± 9.8 50.0 ± 10.1 21.8 ± 8.7

n-3:n-6 n.d. 1.7 6.0 2.9 7.3

individual dry mass at the beginning of each experiment as the covariates. Differences among treatments were analyzed with Tukey’s Honestly Significant Difference (HSD) post hoc test. The dependence of growth rates of C. fluminea on both food components and clam tissue components was assessed by linear regression analyses.

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Results

Elemental and biochemical composition of food sources

Molar C:N and C:P ratios of the cyanobacteria S. elongatus, A. variabilis, and A. flos-aquae and the eukaryotic algae S. obliquus and Cryptomonas sp. were low, results indicating high N and P content (Tab. 1). The lipid composition of cyanobacteria was characterized by high amounts of short-chain saturated (SAFA) and monounsaturated (MUFA) fatty acids and by the absence of sterols. In contrast to A. variabilis and A. flos-aquae, which contained comparatively high amounts of the 2 PUFAs linoleic acid (18:2n-6, LIN) and α-linolenic acid (18:3n-3, ALA), S. elongatus contained no PUFAs. Scenedesmus obliquus contained considerable amounts of ALA and 18:4n-3, and Cryptomonas sp. contained high amounts of the long-chain fatty acid eicosapentaenoic acid (20:5n-3, EPA) and small amounts of docosahexaenoic acid (22:6n-3, DHA) (Tab. 1). Both eukaryotic species were characterized by high total fatty acid, PUFA, and n-3 levels.

Sterols were detected only in the eukaryotic algae. Scenedesmus obliquus contained a total of 9.9 ± 4.2 µg sterol/mg C. Dominant sterols were fungisterol (5α-ergost-7-en-3β-ol; 2.1 ± 0.7 µg/mg C); chondrillasterol ((22E)-5α-poriferasta-7,22-dien-3-ol; 7.4 ± 3.4 µg/mg C); and 22-dihydrochondrillasterol (5α-poriferast-7-en-3β-ol; 0.5 ± 0.1 µg/mg C). In Cryptomonas sp., total sterols averaged 13.4 ± 4.5 µg/mg C and consisted of 2 principal sterols: brassicasterol ((22E)-ergosta-5,22-dien-3β-ol; 4.2 ± 1.4 µg/mg C) and stigmasterol ((22E)-stigmasta-5,22- dien-3β-ol; 9.1 ± 3.0 µg/mg C).

Elemental and biochemical composition of clam tissues

The elemental composition of soft-tissues was characterized by low C:N (4.6 - 5.3) and C:P (~100 - ~140) (Tab. 2), indicating a high N and P content. Nine sterols were detected in clam tissues: cholesterol (cholest-5-en-3β-ol), stigmasterol, brassicasterol, campesterol (campest- 5-en-3β-ol), corbisterol ((22E)-stigmasta-5,7,22E-trien-3β-ol), ergosterol ((22E)-ergosta- 5,7,22-trien-3β-ol), fungisterol, chondrillasterol, and 22-dihydrochondrillasterol (Tab. 2). The total sterol level of field-collected clams at the start of the experiment was 13.8 ± 0.6 µg sterol/mg C. High amounts of the sterols detected in Cryptomonas sp. were found in tissues of clams fed Cryptomonas sp. (Tab. 2), and small amounts of the sterols detected in S.

obliquus were found in tissues of clams fed S. obliquus, results indicating incorporation of dietary phytosterols.

The total FA content of C. fluminea was between 115 and 150 µg/mg C. Highest amounts were measured in field-collected clams at the beginning of the experiment and in individuals fed either of the 2 eukaryotic algae (Tab. 2). Fatty-acid profiles were dominated by saturated fatty acids (SAFAs, 16:0, 18:0, 20:0), monounsaturated fatty acids (MUFAs, 18:1), and PUFAs with ≥18 C atoms, i.e., LIN, ALA, ARA, EPA, docosapentaenoic acid (22:5n-3, DPA), and DHA, but the relative proportion of these fatty acids differed with food sources (Tab. 2).

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Tab. 2 Mean (±1 SD; n = 3 for C:N and C:P, n = 2 for lipids and sterols) elemental and biochemical composition of soft-tissues of Corbicula fluminea fed different cyanobacteria (Synechococcus elongatus, Anabaena variabilis, Aphanizomenon flos-aquae) and eukaryotic algae (Scenedesmus obliquus, Cryptomonas sp.) after 28 d in a laboratory growth experiment. Abbreviations, units, and fatty acid structure are as in Tab. 1. n.d. = below detection limits.

Variable Start S. eolongatus A. variabilis A. flos-aquae S. obliquus Cryptomonas sp.

C:N 4.9 ± 0.2 5.3 ± 0.2 5.0 ± 0.2 4.6 ± 0.0 4.9 ± 0.1 5.3 ± 0.1

C:P 139.2 ± 3.9 122.9 ± 7.5 125.0 ± 0.5 103.3 ± 0.0 131.9 ± 4.5 121.2 ± 3.5

18:2(n-6) 3.9 ± 1.8 n.d. 7.5 ± 1.1 2.9 ± 0.4 8.1 ± 0.2 5.4 ± 0.6

18:3(n-3) 6.8 ± 2.6 3.4 ± 1.2 15.8 ± 2.2 12.7 ± 2.4 17.9 ± 1.3 18.4 ± 1.9

18:4(n-3) 1.8 ± 2.6 n.d. n.d. n.d. 1.1 ± 1.5 4.5 ± 0.6

20:2(n-6) n.d. n.d. 3.2 ± 0.5 n.d. 2.9 ± 0.4 3.9 ± 0.1

20:4(n-6) 10.1 ± 1.3 5.1 ± 0.1 5.7 ± 0.8 7.3 ± 0.3 3.8 ± 1.7 5.6 ± 0.3

20:5(n-3) 9.5 ± 2.00 6.3 ± 1.4 4.6 ± 0.6 5.2 ± 1.1 3.3 ± 0.7 15.7 ± 0.8

22:5(n-3) 8.2 ± 0.2 5.2 ± 0.8 5.0 ± 0.7 6.1 ± 0.4 n.d. 6.3 ± 0.1

22:6(n-3) 17.5 ± 1.3 10.2 ± 1.9 9.1 ± 1.3 11.3 ± 0.3 5.0 ± 1.5 7.0 ± 0.1 Total FA content 149.6 ± 14.7 116.1 ± 25.4 128.8 ± 18.2 115.3 ± 11.7 134.3 ± 20.3 133.8 ± 3.4

SAFA 31.0 ± 4.3 28.8 ± 4.8 24.4 ± 3.5 25.8 ± 3.6 29.9 ± 2.5 29.2 ± 0.2

MUFA 16.6 ± 4.4 19.4 ± 2.2 23.1 ± 3.3 15.4 ± 2.0 27.7 ± 1.6 12.4 ± 0.7

PUFA 57.9 ± 8.8 42.8 ± 5.4 51.0 ± 7.2 45.6 ± 5.0 42.1 ± 1.2 66.7 ± 3.6

n-3 43.9 ± 8.3 25.0 ± 5.3 34.5 ± 4.9 35.4 ± 4.3 27.2 ± 0.7 51.9 ± 3.5

n-6 14.0 ± 0.5 5.1 ± 0.1 16.4 ± 2.3 10.2 ± 0.7 14.9 ± 1.8 14.8 ± 0.2

n-3:n-6 3.1 4.9 2.1 3.5 1.8 3.5

Unidentified sterol 0.7 ± 0.2 0.1 ± 0.2 0.2 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.2

Cholesterol 8.3 ± 1.0 3.4 ± 2.8 4.0 ± 0.4 6.8 ± 0.8 5.7 ± 0.1 7.3 ± 1.1

Brassicasterol 1.8 ± 0.4 0.8 ± 0.6 0.9 ± 0.1 1.6± 0.1 1.4 ± 0.3 9.7 ± 7.1

Ergosterol n.d. n.d. n.d. n.d. 0.2 ± 0.2 1.8 ± 2.5

Campesterol 0.9 ± 0.2 0.3 ± 0.2 0.3 ± 0.0 0.5 ± 0.1 1.7 ± 0.4 0.8 ± 0.6

Stigmasterol 1.2 ± 0.1 0.7 ± 0.5 0.5 ± 0.0 0.9 ± 0.2 3.1 ± 0.8 9.2 ± 7.2

Corbisterol 0.8 ± 0.1 n.d. n.d. n.d. 0.8 ± 0.1 1.3 ± 1.3

Fungisterol 0.1 ± 0.1 n.d. n.d. n.d. 0.5 ± 0.0 n.d.

Chondrillasterol n.d. 0.3 ± 0.4 n.d. n.d. 0.3 ± 0.4 n.d.

22-Dihydrochondrillasterol 0.1 ± 0.2 n.d. n.d. n.d. 0.1 ± 0.1 n.d.

Total sterol content 13.8 ± 0.6 5.7 ± 4.7 5.9 ± 0.6 10.1 ± 1.2 14.0 ± 1.9 30.4 ± 19.8

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Fig. 5 Mean (±1 SD; n = 15) somatic growth rates of Corbicula fluminea fed 5 different food sources for 28 d in a laboratory growth experiment. Bars labeled with different letters differ significantly (Tukey’s Honestly Significant Difference, p < 0.05).

Growth of C. fluminea

In all treatments, the dry mass of C. fluminea increased during the 28 d experiment (positive somatic growth rates; Fig. 5). However, neither the initial mass (ANCOVA, F = 2.96, df = 1, p = 0.09) nor the initial mass × food source interaction (ANCOVA, F =1.30, df = 4, p = 0.28) significantly explained the growth of C. fluminea. Instead, growth rates were significantly affected only by food treatments (ANCOVA, F = 32.9, df = 4, p < 0.001; Tukey’s HSD, p <

0.05). Clams fed the 3 cyanobacteria had lower growth rates than clams fed the 2 eukaryotic algae. Growth rates did not differ significantly among clams fed filamentous (A. variabilis, A.

flos-aquae) or single-celled (S. elongates) cyanobacteria (Fig. 5). Clams fed S. obliquus (0.023

± 0.004/d) and Cryptomonas sp. (0.027 ± 0.004/d) achieved the highest growth rates, but these growth rates were not significantly different.

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Fig. 6 Linear regression models between molar C:N (A) and C:P (B); total fatty acid (FA) (C), total sterol (D), n-3 (E), n-6 (F), saturated (G), monounsaturated (H), and polyunsaturated (I) FA levels; n-3:n-6 ratio (K); and α- linolenic acid (ALA (L) and linoleic acid (LIN) (M) levels in food sources (x-axis) and growth rates of Corbicula fluminea (y-axis). Regression equations are plotted for significant models.

Growth rates of clams were positively correlated with many biochemical components of their food (Fig. 6A–M). For example, C:N (Fig. 6A), total fatty acids (Fig. 6C), total sterols (Fig.

6D), n-3 PUFAs (Fig. 6E), total PUFAs (Fig. 6I), ALA (Fig. 6L), and LIN (Fig. 6M) content of food sources were all significantly positively correlated with clam growth. SAFA (Fig. 6G) and MUFA (Fig. 6H) content of food sources were significantly negatively correlated with growth.

Growth rates of clams (Fig. 7A–M) were significantly positively correlated with total sterols (Fig. 7D), ALA (Fig. 7L), and EPA (Fig. 7M) concentrations in clam soft-tissues.

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Fig. 7 Linear regression models between molar C:N (A) and C:P (B); total fatty acid (FA) (C), total sterol (D), n-3 (E), n-6 (F), saturated (G), monounsaturated (H), and polyunsaturated (I) FA levels; n-3:n-6 ratio (K); and α- linolenic acid (ALA) (L), and eicosapentaenoic acid (EPA) (M) levels in Corbicula fluminea tissue (x-axis) and growth rate of Corbicula fluminea individuals (y-axis). Regression equations are plotted for significant models.

Discussion

Few studies have been done to determine the effects of the food quality of phytoplankton species on freshwater bivalves. Foe and Knight (1986) examined the growth of C. fluminea fed 6 genera of green algae in various combinations. They observed strongly positive tissue growth when clams were fed various mixtures of Ankistrodesmus, Chlamydomonas, Chlorella, and Scenedesmus, but not when they were fed unialgal food sources or mixtures containing Pedinomonas or Selenastrum. However, the nutritional requirements of Corbicula that make some algal species higher-quality food than others have not been further characterized.

In our study, the growth of C. fluminea was significantly affected by the lipid composition of its food. Corbicula fluminea growth rates were significantly lower when juveniles were fed

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cyanobacterial diets than when they were fed eukaryotic algae. This result suggests that cyanobacteria are of poorer food quality than eukaryotic algae for C. fluminea and adds to previous findings that cyanobacteria are a nutritionally inadequate food source for a number of aquatic invertebrates, including D. polymorpha (Wacker et al. 2002; Vanderploeg et al.

2009) or cladocerans of the genus Daphnia (Wilson et al. 2006; Martin-Creuzburg et al.

2008). Cyanobacteria are a poor-quality food for Daphnia because they cannot supply essential lipids, especially sterols (Martin-Creuzburg et al. 2008).

Few studies have been done on the sterol requirements of bivalves, and none have been done on freshwater bivalves. Experiments with marine bivalves suggest that sterols are essential for gametogenesis and for biosynthesis of membranes in the early embryo (Soudant et al. 1996a). Duncan et al. (1987) reported that cholesterol is the main sterol in C.

fluminea with lesser amounts of campesterol, sitosterol, stigmasterol, and brassicasterol.

Recent studies confirm the presence of these sterols in C. fluminea (Chijimatsu et al. 2011).

However, whether these sterols are synthesized de novo by Corbicula or are of dietary origin has not been investigated. The 5 sterols (cholesterol, brassicasterol, campesterol, stigmasterol and corbisterol) we found in C. fluminea agree with those reported by Duncan et al. (1987). In addition, sterols present in the eukaryotic algae S. obliquus and Cryptomonas sp., which were in accordance with former lipid analysis in algae (Martin-Creuzburg et al.

2005b; Martin-Creuzburg et al. 2006), also occurred in Corbicula tissues. Thus, the biochemical composition of clam tissues reflected the biochemical composition of their food sources. However, we cannot determine whether these sterols were assimilated or simply present in the gut of the animals because gut tissues were not dissected prior to analyses.

Total sterol levels in clam tissues decreased relative to initial levels within 4 wk of growth on either of the cyanobacterial diets. In contrast, total sterol levels in clams fed the sterol- containing green alga S. obliquus were constant over the experiment, a result that suggests dietary uptake and possibly homeostatic regulation of sterol levels. Total sterol levels in clams fed Cryptomonas sp. increased during the experiment primarily because of the incorporation of large quantities of dietary brassicasterol and stigmasterol. We cannot show definitively that the correlations we obtained represent causal relationships. Nevertheless, the finding that total sterol levels in the food organisms and in clam tissues were highly correlated with somatic growth rates of the clams suggests that C. fluminea relies on a dietary source of sterols to obtain high growth rates. Hence, growth of clams fed cyanobacterial diets presumably was limited by the absence of sterols, as has been shown for the crustacean Daphnia (Martin-Creuzburg et al. 2008).

PUFAs are another important and well documented class of essential lipids. In particular, molecules with >18 C atoms, such as EPA, play a significant role in animal physiology, and their absence can constrain growth and reproduction of various invertebrate species, such as Daphnia (Müller-Navarra et al. 2000; Wacker and Von Elert 2001) or D. polymorpha (Vanderploeg et al. 1996; Wacker et al. 2002; Wacker and Von Elert 2004). We found strong positive correlations between somatic growth rates and the levels of total PUFAs and n-3

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PUFAs in the diet, which suggests that the growth of C. fluminea is strongly affected by the availability of dietary PUFAs. Moreover, the 2 PUFAs LIN and ALA, which are important precursors for long-chain PUFAs of the n-6 and n-3 series, respectively, also were positively correlated with somatic growth rates of C. fluminea (Fig. 6L, M). Somatic growth rates and the availability of SAFAs and MUFAs in the diet were negatively correlated, results suggesting that growth was not affected by these FAs. Somatic growth rates and tissue levels of ALA and EPA were strongly positively correlated, results suggesting that long-chain PUFAs are of particular importance for growth of C. fluminea. This conclusion is in accordance with studies on nutritional requirements of marine bivalves, which suggest that egg numbers, hatching success, and growth and survival of larvae are closely linked to dietary lipids, particularly to ARA, EPA, and DHA (Soudant et al. 1996a; 1996b; 1996c). Dietary PUFAs also affect egg mass, egg quality, and larval growth of D. polymorpha (Wacker et al. 2002; Wacker and Von Elert 2003; 2004). Hence, in our study, growth of C. fluminea on cyanobacterial diets was possibly constrained by the absence of sterols and, simultaneously, by the absence of long-chain PUFAs. Martin-Creuzburg et al. (2009) used Daphnia as a model organism to show that feeding on a cyanobacterial diet leads to colimitation by sterols and PUFAs. Our data strongly suggest that the growth of juvenile C. fluminea depends on the availability of these essential lipids in the diet. However, we cannot separate the effects mediated by dietary sterols from those mediated by dietary PUFAs (or other potentially colimiting nutrients not considered here) to assess the relative importance of these essential nutrients.

Corbicula fluminea is regarded as a nonselective suspension feeder (Lauritsen 1986; Way et al. 1990; Boltovskoy et al. 1995; Vaughn and Hakenkamp 2001). However, the finding that Anodonta anatina is able to reject filamentous cyanobacteria suggests that size and shape of suspended particles affect particle sorting in bivalves (Ward et al. 1998; Vanderploeg et al.

2001; Bontes et al. 2007). Furthermore, variation in gill surface structures might lead to differences in particle sorting in bivalve species (Payne et al. 1995; Ward et al. 1998; Baker et al. 2000). Chemoreception via recognition of surface structures has been proposed as another mechanism by which bivalves avoid uptake of unsuitable food sources (Espinosa et al. 2010). Vanderploeg et al. (2001; 2009) suggested that D. polymorpha is able to discriminate against large toxic colonies of Microcystis aeruginosa and, thereby, promotes toxic Microcystis blooms. We provided C. fluminea with different phytoplankton species as sole food sources to assess differences in food quality rather than to assess capability for selective feeding. To assess the significance of a lipid limitation in the field, research is needed to test whether C. fluminea can select food particles on the basis of biochemical quality, e.g., sterol or PUFA content.

Preliminary experiments suggested that all food sources used in our study, including the filamentous cyanobacteria, were readily removed from the water column and ingested by C.

fluminea. Moreover, in contrast to starving clams, which did not grow at all, even the clams fed cyanobacterial diets had positive somatic growth rates, which indicated that all offered food sources were of some nutritional use. After 24 h of feeding, significant amounts of food particles were present in the water column, an observation suggesting that clams did not

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reduce food quantity to limiting levels and that a daily supply of 3 mg C/L was sufficient.

Moreover, significant amounts of biodeposited material (feces and pseudofeces) were observed on sediment surfaces in all food treatments. Corbicula fluminea can feed on sedimented or biodeposited organic matter (Boltovskoy et al. 1995; Cahoon and Owen 1996;

Hakenkamp and Palmer 1999). Thus, C limitation in our experiment is rather unlikely.

The biomass-related somatic growth rates obtained in our experiment correspond to an increase in shell length of 0.6 to 1.8 mm over the 28-d experiment (0.02–0.06 mm/d), which is comparable with published data on shell growth of natural C. fluminea populations (Belanger et al. 1990; French and Schloesser 1991; Vohmann et al. 2010).

Molar C:N and C:P of clam tissues were low and comparable to elemental tissue ratios of Corbicula described in the literature (Evans-White et al. 2005). Negative correlations of somatic growth rates with C:N or C:P of the different food sources (i.e., increasing growth rates with decreasing C:N or C:P) would be expected if growth were limited by low availability of N or P. However, in our experiment, somatic growth rates of C. fluminea were positively correlated with dietary C:N, i.e., growth rates increased with a decreasing availability of dietary N. This result together with the finding that the highest growth rates were obtained with algae containing high amounts of lipids but low levels of N suggest that somatic growth of C. fluminea was constrained by a low availability of dietary lipids and not by the availability of dietary N. Corbicula fluminea has a low demand for N (Atkinson et al.

2010) and, therefore, limitation by N in our experimental setup was rather unlikely. Bivalves might bypass elemental nutrient limitations by accumulating N and P within body tissues, a mechanism that could contribute to the success of invasive species (Naddafi et al. 2009).

Moreover, bivalves are involved in nutrient recycling. For instance, bivalves have the potential to affect phytoplankton communities directly by grazing or indirectly via resuspension of limiting nutrients (Arnott and Vanni 1996; Vanni 2002), thereby supporting growth of phytoplankton (Lauritsen 1986; Yamamuro and Koike 1993), which is largely driven by the availability of N and P (Tilman et al. 1982; Elser et al. 1990). Thus, on one hand, bivalve invasion might be influenced by the composition of the phytoplankton community, and on the other hand, bivalve invasion might affect nutrient flows in lake ecosystems.

Annual succession of suspended food organisms (mainly phytoplankton) is associated with changes in food quantity and quality, e.g., changes in the availability of biochemical nutrients for filter-feeding organisms (Wacker and Von Elert 2001), which also may affect the fitness of bivalves. In eutrophic systems, summer months are often characterized by a high proportion of cyanobacteria in the phytoplankton, which may limit availability of essential substances like sterols and PUFAs (Müller-Navarra et al. 2004). The frequency of cyanobacterial bloom formation is expected to increase with global warming (Paerl and Huisman 2008; Wagner and Adrian 2009), and this increased frequency could influence the geographic spread of C. fluminea and other invasive bivalves. However, clams like C.

fluminea might change their feeding mode from seston filtration to deposition feeding.

Increased reliance on benthic food sources could allow clams to outlast possible limitations

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caused by shifts in pelagic food sources (Hakenkamp and Palmer 1999; Nichols and Garling 2000; Raikow and Hamilton 2001; Nichols et al. 2005).

Our finding that the growth of C. fluminea is significantly affected by the availability of dietary sterols and PUFAs contributes to our understanding of how benthic food web processes are affected by biochemical food-quality constraints, a topic that has been studied almost exclusively for pelagic components of the food web. Investigating and comparing the physiological demands of native and invasive species may help us understand invasion patterns and improve risk assessment of upcoming invasions.

Acknowledgements

We thank R. Basen, N. Schlotz and 2 anonymous referees for helpful comments on the manuscript that improved its quality. This work was supported by the DFG (German Research Foundation) within the collaborative research centre SFB 454 “Littoral of Lake Constance”.

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Chapter 3 Absence of sterols constrains food quality of cyanobacteria for an invasive freshwater bivalve

Timo Basen, Karl-Otto Rothhaupt and Dominik Martin-Creuzburg

Oecologia

doi:10.1007/s00442-012-2294-z received: 28. September 2011 accepted: 22. February 2012

Abstract

The accumulation of cyanobacterial biomass may severely affect the performance of aquatic consumers. Here, we investigated the role of sterols in determining the food quality of cyanobacteria for the invasive clam Corbicula fluminea, which has become a common benthic invertebrate in many freshwater ecosystems throughout the world. In standardized growth experiments, juvenile clams were fed mixtures of different cyanobacteria (Anabaena variabilis, Aphanothece clathrata, Synechococcus elongatus) or sterol-containing eukaryotic algae (Cryptomonas sp., Nannochloropsis limnetica, Scenedesmus obliquus). In addition, the cyanobacterial food was supplemented with different sterols. We provide evidence that somatic growth of C. fluminea on cyanobacterial diets is constrained by the absence of sterols, as indicated by a growth-enhancing effect of sterol supplementation. Thus, our findings contribute to our understanding of the consequences of cyanobacterial mass developments for benthic consumers and highlight the importance of considering sterols as potentially limiting nutrients in aquatic food webs.

Key words: benthic-pelagic coupling, Corbicula fluminea, cyanobacterial blooms, essential lipids, food quality

Nutritional aspects in the invasive freshwater bivalve Corbicula fluminea : The role of essential lipids