Phytoplankton food quality effects on gammarids: benthic–pelagic coupling mediated by an invasive freshwater clam
Timo Basen, Rene Gergs, Karl-Otto Rothhaupt, and Dominik Martin-Creuzburg
Abstract:Benthic–pelagic coupling mediated by bivalves has been shown to increase the flow of energy towards the benthos. To assess the capability of clams to process and therewith modify the quality of pelagic food sources for subsequent use by benthic invertebrates, we conducted a growth experiment in which juvenileGammarus roeseliiwere raised either directly on sedimented pelagic autotrophs (algae, cyanobacteria) or on the same autotrophs biodeposited by the invasive freshwater clamCorbicula flumineaeither as feces or pseudofeces. We show that growth and survival ofG. roeseliiare significantly improved when autotrophs are offered as biodeposition material and suggest that this clam-mediated upgrading of food quality is due to both an increased bioavailability of pelagic food particles, which are packed in mucus during clam processing, and an increased dietary provisioning with essential lipids (sterols and (or) polyunsaturated fatty acids) originating from the clams. Hence, filter-feeding bivalves provide a crucial link between the pelagic and benthic food web not only by deflecting energy fluxes, but also by processing and upgrading pelagic food (increased bioavailability, improved biochemical composition) for benthic invertebrates.
Résumé :Il a été démontré que le couplage benthique-pélagique par l'entremise de bivalves accroît le transfert d'énergie vers le benthos. Afin d'évaluer la capacité des palourdes de transformer des sources de nourriture pélagiques et d'en modifier ainsi la qualité pour utilisation subséquente par des invertébrés benthiques, une expérience de croissance a été menée dans laquelle desGammarus roeseliijuvéniles ont été élevés directement sur des autotrophes (algues, cyanobactéries) pélagiques déposés ou sur les mêmes autotrophes biodéposés comme fèces ou pseudofèces par la palourde d'eau douce envahissante Corbicula fluminea. Nous démontrons des améliorations significatives de la croissance et de la survie de G. roeseliiquand des autotrophes sont offerts sous forme de matières biodéposées et postulons que cette amélioration de la qualité de la nourriture par l'entremise des palourdes est due a` la biodisponibilité accrue des particules de nourriture pélagiques, qui sont emmagasinées dans du mucus durant l'assimilation par la palourde, ainsi qu'a` un apport alimentaire accru de lipides essentiels (stérols ou acides gras polyinsaturés) provenant des palourdes. Ainsi, les bivalves filtreurs constituent un lien clé entre les réseaux trophiques pélagique et benthique non seulement en détournant les flux d'énergie, mais également en traitant et en améliorant la nourriture pélagique (biodisponibilité accrue, composition biochimique améliorée) pour les invertébrés benthiques. [Traduit par la Rédaction]
Introduction
Geographical spread of invasive species is recognized as a main cause of the omnipresent decline of freshwater biodiversity (Sala et al. 2000). In the past century, the Asian clamCorbicula fluminea has become an ubiquitous benthic invertebrate in freshwater eco- systems worldwide (Araujo et al. 1993;Darrigran 2002;Lee et al.
2005). Filter-feeding bivalves can considerably increase the pelagic–benthic coupling (i.e., the flow of pelagic organic matter to the benthos), thereby stimulating benthic productivity (Strayer et al. 1999;Sousa et al. 2008;Gergs et al. 2009). The occurrence of Corbiculapopulations has also been shown to lead to an increase in sediment organic matter concentrations (Hakenkamp and Palmer 1999). The biodeposited material mainly consists of digested (feces) and undigested, rejected (pseudofeces, PSF) seston particles.
At low particle concentrations, the deposited material consists mainly of feces (MacIsaac and Rocha 1995; Roditi et al. 1997), whereas with rising food concentrations (>0.2 mg·L−1of carbon for Dreissena polymorpha), the proportion of PSF increases (Walz 1978; Gergs et al. 2009). For many benthic invertebrates, these bivalve-generated food packages represent a suitable food source (Karatayev et al. 1997;Roditi et al. 1997). Biodeposition material of bivalves has also been shown to support gammarid nutrition both
in the laboratory and in the field (Gergs and Rothhaupt 2008;
Gergs et al. 2011).
Besides bivalve-driven deposition of organic matter, sedimenta- tion of phytoplankton per se, especially under bloom conditions, provides a huge pelagic carbon pool for the benthic food web (Nascimento et al. 2008;Suikkanen et al. 2010). Benthic inverte- brates can ingest and assimilate sedimented cyanobacteria, but the nutritional value seems to be rather low (Karlson et al. 2008;
Nascimento et al. 2009). As the frequency of cyanobacterial bloom formation is expected to increase with global warming (Paerl and Huisman 2009), it is important to investigate the consequences of cyanobacterial mass developments for ecosystem processes (e.g., the role of cyanobacterial carbon within the food web and its food quality for pelagic and benthic consumers). In general, cyanobacteria represent a nutritionally inadequate food source for aquatic consum- ers, possibly owing to morphological properties (Van Donk et al.
2011), toxin production (DeMott et al. 1991), and (or) a deficiency in essential biochemical nutrients (Müller-Navarra et al. 2000;
Martin-Creuzburg et al. 2008). In particular, the lack of sterols has been suggested to constrain the carbon transfer efficiency from cya- nobacteria to herbivorous zooplankton and benthic filter feeders (Martin-Creuzburg et al. 2008;Basen et al. 2011,2012).
T. Basen, K.-O. Rothhaupt, and D. Martin-Creuzburg.Limnological Institute, University of Constance, Mainaustrasse 252, 78464 Konstanz, Germany.
R. Gergs.Institute for Environmental Sciences, University of Koblenz-Landau, Fortstrasse 7, 76829 Landau, Germany.
Corresponding author:Timo Basen (e-mail:timobasen@googlemail.com).
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http://dx.doi.org/10.1139/cjfas-2012-0188
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-245453
Although several studies have shown that bivalves are signifi- cantly involved in transferring pelagic organic matter (i.e., mainly phytoplankton) to the benthic food web (e.g.,Gergs et al. 2009), the quality of this biodeposited material as food for benthic inver- tebrates has been poorly studied. In the present study, we inves- tigated whether biodeposition materials produced byC. fluminea feeding on different pelagic food sources differ in their food qual- ity forGammarus roeselii. We hypothesized that the biodeposition activity does not only provide increased access to pelagic food sources, but also affects the nutritional quality of phytoplankton as food for benthic invertebrates by modifying the elemental and biochemical composition of the diet. In laboratory experiments, two different concentrations of algae (Nannochloropsis limnetica, Scenedesmus obliquus) and cyanobacteria (Anabaena variabilis, Syn- echococcus elongatus) were fed to adultC. flumineato obtain biode- position material consisting of either mostly digested (feces) or undigested (PSF) material. We investigated the survival and growth ofG. roeseliifeeding on collected biodeposition materials and on sedimented autotrophic food sources without clam condi- tioning, and related the results to the elemental (carbon, nitrogen, phosphorus) and biochemical (fatty acid, sterol) compo- sition of the different food sources to assess the role of clam filtration and digestion in determining the nutritional quality of phytoplankton-derived food for benthic invertebrates.
Materials and methods
Sampling and cultivation of animals
Gammarids (G. roeselii) and clams (C. fluminea) were obtained from the littoral zone of the oligotrophic prealpine Lake Con- stance. AdultG. roeseliiwere collected via kick sampling at the shoreline, and clams were collected at a water depth of 2–3 m by scuba diving. Until the start of the experiments, both species were kept separately in climate chambers with a diurnal dark–light cycle of 12 h : 12 h.Gammarus roeseliiwere kept at 15 °C in aerated aquaria containing lake water, gravel of different grain sizes for shelter, and dried alder leaves as a food source;C. flumineawere kept at 20 °C in a flow-through system with seston-containing lake water (<30m) and washed sediment.
Food preparation
Autotrophic food sources were cultivated semicontinuously in aerated 5 L glass bottles at a dilution rate of 0.20 day−1at 20 °C with illumination at 100–120mol photons·m−2·s−1and harvested in the late-exponential growth phase. The coccoid cyanobacterium Synechococcus elongatus(SAG 89.70, Sammlung für Algenkulturen Göttingen, Germany), the filamentous cyanobacteriumAnabaena variabilis(ATCC 29413, American Type Culture Collection, Manas- sas, Virginia, USA), the green algaScenedesmus obliquus(SAG 276- 3a), and the eustigmatophyteNannochloropsis limnetica(SAG 18.99) were each grown in Cyano medium (Jüttner et al. 1983). These nontoxic food organisms were used because they differ consider- ably in size, shape, lipid content, and composition and have been shown previously to differ in food quality for freshwater inverte- brates (Martin-Creuzburg et al. 2008;Basen et al. 2012). Food sus- pensions were prepared by concentrating the cells via centrifugation (3000g, 10 min) and resuspension in fresh medium. Carbon (C) con- centrations of the food suspensions were estimated from photo- metric light extinctions (800 nm) and from carbon-extinction equations determined prior to the experiment.
To obtain biodeposition material produced by clams,C. fluminea were kept in flow-through systems with filtered, aerated lake wa- ter (<1m, 200 mL·min−1, 20° C) enriched with cyanobacteria or algae. The flow-through systems consisted of experimental basins with a size of 34 cm × 40 cm × 7.5 cm (width × depth × height); the water level was adjusted to 6 cm, resulting in a water volume of approximately 8 L. To minimize sedimentation of the algae, the water in the basins was gently aerated. All flow-through systems
were equipped with small plastic boxes (8 cm × 8 cm × 5 cm;n= 5 per basin) containing approximately 12 g of clam wet mass bio- mass (shell length 10–20 mm). The autotroph food suspensions were added continuously from reservoirs containing daily re- newed food suspensions using a peristaltic pump; the food con- centrations were constant throughout the experiment. Two different food concentrations were adjusted to produce two dif- ferent kinds of biodeposition material: a low food concentration (0.2 mg C·L−1) was used to gain mostly digested autotrophic carbon (henceforth referred to as feces), and a high food concentration (1 mg C·L−1) was used to increase the fraction of undigested au- totrophic carbon (henceforth referred to as PSF). Clam-conditioned feces and PSF differed in color and thus could be easily distinguished.
The organic matter biodeposited byC. flumineain the plastic boxes was collected three times a week (i.e., the plastic boxes were replaced by clean boxes), and the biodeposited material was rinsed from the boxes using a pipette, centrifuged, resuspended in filtered lake wa- ter, and adjusted to a certain optical density to ensure constant feed- ing conditions forG. roeselii. Preliminary experiments revealed that food suspensions prepared according to this procedure are highly suitable to provideG. roeseliiwith well-defined food concentrations.
Analyses of food sources
The elemental (carbon, nitrogen, phosphorus) and biochemical (fatty acids, sterols) composition of the food sources were deter- mined weekly from aliquots of the food suspensions. The num- bers of replicates differed among treatments (3–7) because the collected biodeposited material was scarce and preferentially fed to amphipods (i.e., only the remaining material was used for bio- chemical analyses). Aliquots were filtered onto precombusted glass-fiber filters (Whatman GF/F, 25 mm diameter) and analyzed for particulate organic carbon (POC) and nitrogen using an NCS-2500 analyzer (ThermoQuest). For the determination of particulate phosphorus, aliquots were collected on acid-rinsed polysulfone filters (HT-200; Pall) and digested with a solution of 10% potassium peroxodisulfate and 1.5% sodium hydroxide for 60 min at 121 °C. Soluble reactive phosphorus was determined using the molybdate – ascorbic acid method (Greenberg et al.
1985). Values are expressed as molar carbon to nitrogen (C:N) and molar carbon to phosphorus ratios (C:P).
Lipids were extracted two times from precombusted GF/F filters (Whatman, 25 mm diameter) loaded with approximately 0.5 mg (for fatty acid analysis) or 1 mg (for sterol analysis) POC of the food suspensions using a mixture of dichloromethane–methanol (2:1,v/v). For the analysis of sterols, the pooled cell-free extracts were dried under a stream of pure gaseous N2and saponified with 0.2 mol·L−1methanolic KOH (70 °C, 1 h). Subsequently, sterols were partitioned into isohexane – diethyl ether (9:1,v/v), again dried under a stream of N2, and resuspended in a volume of 20L isohexane. For the analysis of fatty acids, the cell-free extracts were dried under a stream of N2and esterified with 3 mol·L−1 methanolic HCl (60 °C, 20 min). Subsequently, fatty acid methyl esters (FAMEs) were partitioned into isohexane, dried under a stream of N2, and resuspended in a volume of 50L isohexane.
Lipids were analyzed by gas chromatography on an HP 6890 gas chromatograph (Agilent Technologies) equipped with a flame ion- ization detector and either a DB-225 (J&W Scientific) capillary col- umn to analyze FAMEs or an HP-5 (Agilent Technologies) capillary column to analyze sterols. Details of gas chromatography config- urations are given elsewhere (Martin-Creuzburg et al. 2009,2010).
Lipids were quantified by comparison to internal standards (C17:0 and C23:0 methyl esters; 5␣-cholestan). The detection limit was
⬃20 ng of sterol or fatty acid. Lipids were identified by their re- tention times and their mass spectra, which were recorded with a gas chromatograph – mass spectrometer (Finnigan MAT GCQ) equipped with a fused silica 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 EI ionization mode, and lipids were identified by comparison with mass spectra of reference substances purchased from Sigma or Steraloids and (or) mass spectra found in a self-generated spectra library or in the literature (e.g.,Goad and Akihisa 1997). The abso- lute amount of each lipid was related to POC.
Growth experiments withGammarus roeselii
Gammarids were maintained in tanks filled with water from Lake Constance containing gravels of different grain sizes for shel- ter. Gammarids were fed on dried alder leaves. For the growth experiments, juvenile gammarids (2–3 mm body length) hatched in these tanks were used. Gammarid growth experiments were conducted from June to October 2009 in glass beakers filled with 100 mL of filtered lake water (<1m); a small stone (organic mat- ter removed using a muffle furnace) was provided as shelter in each beaker. JuvenileG. roeseliiwere randomly transferred to the experimental beakers. The experiment comprised the following food treatments: (1) the two cyanobacteria — S. elongatus and A. variabilis; (2) the two eukaryotic algae—S. obliquusandN. lim- netica; or (3) the same food organisms as the biodeposited material that is produced by feeding the clam with either low or high concentrations of these food organisms (i.e., feces or PSF). The food suspensions were prepared and renewed daily. Gammarids were fed ad libitum (Gergs and Rothhaupt 2008) or starved with- out adding food (starvation as a control treatment). Food concen- trations were adjusted to the actual body mass the gammarids achieved during the experiment, estimated using a previously established body length – dry mass regression (Baumgärtner and Rothhaupt 2003). To avoid a limitation by food quantity, one-third of the total body mass (mg C) was provided as food per individual per day, as suggested byGergs and Rothhaupt (2008). Each food treatment was replicated 20 times; each replicate consisted of one individual, and individuals were monitored for 7 weeks. Three times a week gammarids were transferred into new beakers to avoid the accumulation of food and fecal pellets and the forma- tion of biofilm. Body lengths of the gammarids were measured once a week as described in Gergs and Rothhaupt (2008), and survival was recorded three times a week.
Statistical analyses
All results were statistically analyzed using the statistical soft- ware package R (R Development Core Team 2006). Differences between food species and food treatments in total sterol and total polyunsaturated fatty acid (PUFA) concentrations (g·(mg C)−1), and molar C:N and C:P ratios, respectively, were analyzed using analysis of variance (ANOVA) followed by Tukey's post hoc tests separately for the different food species and food treatments. Data
were square-root transformed (sterols, C:N, C:P) and ln(x+ 2)- transformed (PUFAs) to obtain homogeneity of variances (Levene's test).
The survival of gammarids in the growth experiments was an- alyzed as the dependent variable, with the offered food species (A. variabilis,N. limnetica,S. elongatus, andS. obliquus) and the type of the food treatment (i.e., autotrophs, feces and PSF) serving as the independent variables. We used the parametric survival model
“survreg” fitted to an exponential data distribution (␣= 0.05).
Comparisons between treatments were done using general linear hypotheses and multiple comparisons with Tukey's post hoc test for parametric models. For the two-way analyses, model simplifi- cation was performed stepwise. Model comparison was done us- ing ANOVA with a2test; significant differences from the full model indicate a loss of information through model simplifica- tion. Differences in body length at the end of the growth experi- ment were analyzed using ANOVA followed by Tukey's post hoc tests, which were performed separately for the different food spe- cies and food treatments. A Student's ttest was performed in those treatments where only two gammarids survived. Homoge- neity of variances was tested using Bartlett's test.
Results
Analyses of food sources Food stoichiometry
In all three food treatments the cyanobacteria had lower molar C:N ratios compared with the eukaryotic algae (Table 1). Nitrogen levels were reduced in feces produced on cyanobacterial diets, leading to a significant increase in C:N ratios (S. elongatus: p = 0.004;A. variabilis:p< 0.001). C:N ratios of the algaN. limneticawere significantly lower than those of the biodeposition materials pro- duced by C. flumineafeeding onN. limnetica(Table 1; Fig. 1). In contrast, C:N ratios of the green algaS. obliquus did not differ significantly from C:N ratios ofS. obliquus-based biodeposition ma- terials (p= 0.06). The molar C:P ratios showed no significant dif- ferences between food treatments for any of the cyanobacteria or algae used. However, C:P ratios of PSF and feces produced on an S. elongatusdiet were lower than C:P ratios of PSF and feces pro- duced on the other autotrophs (Table 1).
Fatty acid composition
In the cyanobacteriumS. elongatus, only saturated (14:0, 16:0) and monounsaturated fatty acids (16:1, 18:1) were detected. In con- trast, small amounts of the PUFAs 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) were detected in feces of Table 1.Statistical analysis of molar carbon to nitrogen (C:N) ratios, carbon to phosporus (C:P) ratios, total
polyunsaturated fatty acids (PUFAs), and total sterol contents of the different food sources used to raise Gammarus roeselii.
Species Treatment Effect df C:N C:P PUFAs Sterols
Two-way analysis
All All Species 3 <0.001 <0.001 <0.001 <0.001
Treatment 2 <0.001 0.46 0.03 0.03
Species × treatment 6 0.02 0.03 0.26 0.03
One-way ANOVA and Tukey's HSD
S. elongatus All Treatment 2 0.004 0.08 — <0.001
A. variabilis 2 <0.001 0.41 0.07 0.10
S. obliquus 2 0.06 0.09 0.19 0.21
N. limnetica 2 <0.001 0.22 0.004 0.97
One-way ANOVA and Tukey's HSD
All Autotrophs Species 3 <0.001 0.14 <0.001 <0.001
Feces 3 <0.001 0.002 0.004 0.005
PSF 3 0.01 0.003 <0.001 0.27
Note:Significant results are in bold.
Fig.1.Molarcarbontonitrogen(C:N)andcarbontophosphorus(C:P)ratiosofthedifferentfoodsourcesusedtoraiseGammarusroeselii.Thetwocyanobacteria(S.elongatusand A.variabilis)andthetwoeukaryoticalgae(S.obliquusandN.limnetica)wereeitherfeddirectlytoG.roeselii(autotrophs,whitebars)orafterpassagethroughtheclamC.fluminea(i.e.,either asfeces(hatchedbars)oraspseudofeces(PSF,graybars)).ThedifferentbiodepositionmaterialswereproducedbyfeedingC.flumineawithlow(feces)andhigh(PSF)autotrophconcentrations. Dataareshownasmeansandstandarddeviations;numbersatthebottomofthebarsindicatesamplesizes.StatisticalanalyseswereperformedseparateforC:NandC:Pratiosinallfour foodspecies.Barslabeledwiththesamelettersarenotsignificantlydifferent,andn.s.representsnonsignificantdifferences.
Fig.2.Totalpolyunsaturatedfattyacids(PUFAs)andtotalsterollevels(g·mgC−1)ofthedifferentfoodsourcesusedtoraiseGammarusroeselii.Thetwocyanobacteria(S.elongatusand A.variabilis)andthetwoeukaryoticalgae(S.obliquusandN.limnetica)wereeitherfeddirectlytoG.roeselii(autotrophs,whitebars)orafterpassagethroughtheclamC.fluminea(i.e.,either asfeces(hatchedbars)oraspseudofeces(PSF,graybars)).ThedifferentbiodepositionmaterialswereproducedbyfeedingC.flumineawithlow(feces)andhigh(PSF)autotrophconcentrations. Dataareshownasmeansandstandarddeviations;numbersatthebottomofthebarsindicatesamplesizes.StatisticalanalyseswereperformedseparatefortotalPUFAsandtotalsterol levelsinallfourfoodspecies.Barslabeledwiththesamelettersarenotsignificantlydifferent,andn.s.representsnonsignificantdifferences.
C. flumineawhile feeding onS. elongatus(Table S11). The filamen- tous cyanobacterium A. variabilis contained two PUFAs, 18:2n-6 and 18:3n-3 (␣-linolenic acid, ALA), which were also detected in feces and PSF produced byC. flumineawhile feeding on this cya- nobacterium. In the green algaS. obliquus, four C-18 PUFAs were detected (18:2n-6, 18:3n-6, ALA, 18:4n-3), but no PUFA with more than 18 carbon atoms.Nannochloropsis limneticacontained the high- est amounts of PUFAs; the principal fatty acid was EPA. Besides EPA, also ALA, ARA, and 18:4n-3 were present in allN. limnetica- based food treatments; traces of 22:6n-3 (docosahexaenoic acid;
DHA) were additionally detected in PSF. In all biodeposition ma- terials (feces and PSF), the composition of fatty acids did not differ from that of the respective autotrophs. However, total PUFA levels were reduced in biodeposited material compared with those in cyanobacteria and algae and were significant only forN. limnetica- based diets (N. limnetica:p= 0.003;Table 1;Fig. 2). Among all au- totrophic species, the highest PUFA concentration was found in N. limnetica. The total fatty acid composition and the concentra- tions of single fatty acids in the different food sources are pre- sented in Supplementary Table S11. Without food addition, water samples taken in the flow-through systems contained, on average, 0.2 ± 0.0 mg C·L−1, providing small amounts of fatty acids (total fatty acids: 22.8 ± 6.9g·L−1: respectively, 52.1 ± 15.9g·(mg C)−1for the low food concentration (i.e., feces) and 18.4 ± 5.6g·(mg C)−1 for the high food concentration (i.e., PSF)). The small amounts of fatty acids detected in the water samples consisted of 16:0, 16:1, ALA, and EPA.
Sterol composition
Sterols were not detected in the two cyanobacteria studied (A. variabilisandS. elongatus). However, sterols were detected in biodeposition material produced byC. flumineathat were feeding on the cyanobacteria and consisted of cholesterol (cholest-5-en- 3-ol), sitosterol (stigmast-5-en-3-ol) and stigmasterol ((22E)- stigmasta-5,22-dien-3-ol) (Fig. 2; Table S11). The green alga S. obliquuscontained fungisterol (5␣-ergost-7-en-3-ol), chondril- lasterol ((22E)-5␣-poriferasta-7,22-dien-3-ol), and schottenol (5␣- stigmast-7-en-3-ol). Principal sterols in N. limnetica were cholesterol, sitosterol, and isofucosterol ((24Z)-stigmasta-5,24(28)- dien–3-ol). The sterols detected in biodeposition materials did not differ from those detected in the corresponding algal food sources, neither in composition nor in quantity (S. obliquus:
p= 0.21;N. limnetica:p= 0.97;Fig. 2;Tables 1and S11). In water
samples taken in the flow-through systems, small amounts of sterols were also detected (total sterols: 0.6g·L−1; 2.5 ± 0.6g·(mg C)−1 for feces and 0.9 ± 0.2g·(mg C)−1of carbon for PSF), with choles- terol (1.4 ± 0.3g·(mg C)−1for feces and 0 5 ± 0.1g·(mg C)−1for PSF) as a major component.
Gammarid growth and survival experiment
Without food addition, all individuals died within 1 week. Thus, all survival and growth effects can be attributed to the offered food sources. Survival of gammarids on the different food sources was affected by both the food treatment (i.e., feces, PSF, and with- out clam conditioning) and the autotrophic species as well as by the interactions of these factors (p< 0.01;Table 2). Model simpli- fication resulted in a significant loss of information (p < 0.01) (Table 2), and thus the full model without simplification was used.
With regard to theS. elongatus-based diets,G. roeseliisurvival was higher when fed digestedS. elongatus(i.e., feces) than when fed sedimentedS. elongatusor when fedS. elongatusbiodeposited as PSF (Table 2;Fig. 3;p< 0.001). Survival rates ofG. roeseliidid not differ significantly among theA. variabilis-based diets (p= 0.52).
With both theS. obliquus- and theN. limnetica-based diets, survival ofG. roeseliiwas lowest when sedimented algae were provided, intermediate when packed as PSF, and highest when packed as feces, although the differences between PSF and feces were not statistically significant (Table 2;Fig. 3). Interspecific comparison among food sources revealed no significant differences between survival rates of gammarids, neither for autotrophs nor for feces (Table 2). PSF produced byC. fluminea while it was feeding on S. elongatusresulted in lower survival ofG. roeseliithan PSF based on eukaryotic algal diets. PSF based onA. variabilisdid not differ from all other PSF diets.
During the 7-week experiment, the body length ofG. roeselii increased under all food treatments. Differences in body length among food treatments were unnoticeable prior to 3 weeks of growth (Fig. 3). The starved individuals in the control treatment died within the first week of the experiment; body growth of these animals was not evaluated. Overall, the increase in body length was less pronounced on S. elongatus-based diets than on diets based on the eukaryotic algae (p< 0.001;Table 3;Fig. 3). Further- more, feces and PSF, irrespective of whether they were produced on cyanobacterial or on algal diets, resulted in higher growth rates ofG. roeseliithan that of sedimented cyanobacteria or algae (p< 0.001). When fedS. elongatus-based diets, the final body length
1Supplementary data are available with the article through the journal Web site athttp://nrcresearchpress.com/doi/suppl/10.1139/cjfas-2012-0188.
Table 2.Survival ofGammarus roeseliianalyzed using a parametric survival regression model (survreg) and multiple comparisons followed by Tukey's test for parametric models.
2 df pvalue Residual diff.
Species × treatment 75.16 11 <0.001 1065.7 Species + treatment 59.04 5 <0.001 1081.8*
Species 5.37 3 0.15 1135.5*
Treatment 52.95 2 <0.01 1087.9*
2 df pvalue Autotrophs Feces PSF
S. elongatus 34.62 2 <0.001* a b a
A. variabilis 1.29 2 0.52 a a a
S. obliquus 23.10 2 <0.001* a b b
N. limnetica 10.79 2 0.005* a b ab
2 df pvalue S. elongatus A. variabilis S. obliquus N. limnetica
Autotrophs 4.55 3 0.21 a a a a
Feces 7.83 3 0.05 a a a a
PSF 9.83 3 0.02* a ab b b
Note:Tested models were compared with the full model by ANOVA using the2test. Model simplification resulted in a significant loss of information (p< 0.01), and thus the full model without simplification was used. Different letters indicate significant differences between treatments (Tukey's HSD,p< 0.05).
Fig.3.GrowthandsurvivalofGammarusroeseliifeddifferentcyanobacteria(S.elongatus,A.variabilis)andeukaryoticalgae(S.obliquus,N.limnetica)eitherdirectly(opencircles)orafter passagethroughtheclamC.fluminea(i.e.,eitherasfeces(opentriangles)oraspseudofeces(PSF,greysquares)).ThedifferentbiodepositionmaterialswereproducedbyfeedingC.fluminea withlow(feces)andhigh(PSF)autotrophconcentrations.Differencesbetweenfoodtreatmentsinfinallengthorsurvivinggammaridswereanalyzedseparatelyforeachalgaespecies. Differentlettersindicatesignificantdifferencesbetweentreatments(p<0.05).
ofG. roeseliicould be determined only on feces, as all individuals died in the two other food treatments during the experiment.
When fedA. variabilis-based diets, the increase in body length was significantly less pronounced on the sedimented cyanobacterium than on A. variabilis-containing feces or PSF (p < 0.001). Final lengths of gammarids raised on the different N. limnetica- or S. obliquus-based diets differed significantly, with higher values obtained on PSF than on feces or sedimented algae. No gammarid survived on a sedimentedS. obliquusdiet and thus the final body length could not be determined (Table 3;Fig. 3). The final body lengths of gammarids raised on the cyanobacteriumA. variabilis were significantly lower than the body lengths of gammarids raised on the eukaryotic algaN. limnetica(p= 0.036). Final body lengths ofG. roeseliiwere significantly lower when fedS. elongatus- based feces than when fed the other autotroph-based feces. Apart from that, body lengths did not differ significantly among the different kinds of feces offered toG. roeselii. The final body lengths of G. roeseliifed N. limnetica-based PSF was higher than that of gammarids fedA. variabilis- orS. obliquus-based PSF; gammarids fed S. elongatus-based PSF did not survive the experiment (Table 3;
Fig. 3).
Discussion
Benthic–pelagic coupling mediated by bivalves has been shown to quantitatively affect the flux of energy from pelagic sources to the benthic food web (Newell 2004). However, little is known regarding whether this bivalve-mediated coupling also qualita- tively improves phytoplankton-derived carbon for benthic inver- tebrates in comparison to direct sedimentation of phytoplankton.
Our results indicate that biodeposition of organic matter by bi- valves not only shifts pelagic resources to the benthos (e.g.,Gergs et al. 2009), but also increases the nutritional value of pelagic autotrophs as food for a benthic invertebrate. We show that sur- vival and somatic growth of the gammaridG. roeseliiis enhanced when it feeds on biodeposition material produced by the clam C. flumineaas compared with when it feeds directly on sedimented pelagic algae or cyanobacteria.
In our laboratory study, growth and survival of gammarids was lowest when they were raised directly on sedimented cya- nobacteria or algae (i.e., without passage through the clam). How- ever, G. roeseliiwas able to grow and survive the experimental period of 7 weeks with sedimented autotrophs as the sole food source; this was the case for the filamentous cyanobacterium A. variabilisand the eukaryotic algaN. limnetica. Compared with starved animals, all of which died within the first days of the
experiment, survival of gammarids was also extended when they were provided withS. elongatusorS. obliquus, indicating that the animals were able to use these food sources to some extent. This suggests that freshwater benthic invertebrates benefit from sedi- mented pelagic carbon sources and that the intensity of this ben- eficial effect depends on the phytoplankton species composition.
Survival of G. roeseliiwas highest on clam feces in all four au- totroph treatments, followed by survival on pseudofeces; survival was lowest on sedimented autotrophs. The observed clam- mediated food quality improvement might be due to packing of food items into larger particles during passage through the clam (Atkinson et al. 2011). Filtered autotroph cells are concentrated in clam-born mucus prior to their release as feces or PSF, and these
“packets” of food might be more easily accessible forG. roeselii than the unconditioned autotroph cells. Moreover, besides trap- ping particles from the water column, clam-born mucus itself might also be of nutritional value to gammarids, as the mucus harbors and potentially supports growth of microorganisms (Connor and Quinn 1984;Davies et al. 1992;Guo et al. 2009). Dif- ferences in food quality between feces and PSF might be due to a different constitution of deposited organic matter. When food particles are ingested and at least partly digested by the clams, changes in their size or stability may improve the efficiency of ingestion or digestion by deposition feeders, potentially explain- ing differences in food quality between feces and PSF, as was found in our study.
The qualitative improvement of pelagic carbon by the clams was most evident whenG. roeseliiwas fedS. elongatus-based diets, as growth and survival ofG. roeselii was possible only on feces produced by clams feeding onS. elongatus. In contrast, diets based on the filamentous cyanobacteriumA. variabilisdid not differ in their effects on survival rates of G. roeselii. We propose that these differences in food quality between the two cya- nobacteria are based on morphological differences between single-celledS. elongatus and colony-forming A. variabilis. How- ever, the final body length ofG. roeseliiwas higher in animals fed A. variabilis-based biodeposition materials (i.e., feces and PSF) than in animals raised directly onA. variabilis. The structural benefit of clam biodeposited material forG. roeseliiis not evident for colony- forming autotrophs, as gammarids were able to ingest even sedi- mentedA. variabilis. Our data imply that the observed nutritional upgrading of cyanobacterial food mediated by C. flumineais at least partially due to an enrichment of the biodeposited material with biochemical nutrients during passage through the clam.
Thus, the traces of sterols and PUFAs detected inS. elongatus-based Table 3.Two-way analyses of final lengths of survivingGammarus roeselii, with food species (i.e.,S. elongatus,
A. variabilis,S. obliquus,N. limnetica) and food treatments (i.e. autotroph, feces, PSF) as independent factors.
F df pvalue Tukey's HSD post hoc test
Species 7.68 2 <0.001 S. elongatus(a),A. variabilis(ab), S. obliquus(b),N. limnetica(b);
Treatment 35.60 3 <0.001 Autotrophs (a), feces (b), PSF (b) Species × treatment 7.74 4 <0.001
F df p value Autotrophs Feces PSF
S. elongatus — — — — a —
A. variabilis 14.79 2 <0.001 a b b
S. obliquus t= 2,13 — <0.049 — a b
N. limnetica 22.62 2 <0.001 a b c
F df p value S. elongatus A. variabilis S. obliquus N. limnetica
Autotrophs t= –2.98 — 0.036 — a — b
Feces 7.28 3 <0.001 a b b ab
PSF 10.66 3 <0.001 — a a b
Note:Differences between food treatments were analysed separately for each autotroph species and differences between autotroph species were analysed separately for each food treatment using Tukey's HSD tests. In cases where only two gammarids survived, a Student'sttest was performed. Different letters indicate significant differences between treatments, withp< 0.05 in bold.
biodeposits, in particular in feces, may have increased the proba- bility of survival and may have allowed for at least moderate growth ofG. roeselii. Biodeposition material based onA. variabilis did not affect survival but significantly improved growth of G. roeseliias compared with sedimentedA. variabilis, suggesting that the increased bioavailability of the sedimentedA. variabilis filaments supported survival (in contrast to sedimented single- celledS. elongatus) but not growth (similar to sedimentedS. elonga- tus) ofG. roeseliiand that the observed growth improvement on both S. elongatus- and A. variabilis-based biodeposition materials was due to the increased availability of sterols. PUFAs did not increase in theA. variabilis-based biodeposition materials com- pared with levels in sedimentedA. variabilisand thus are unlikely to be responsible for the observed growth improvement on A. variabilis-based biodeposition material. This observation adds to previous findings showing that the growth of aquatic invertebrates (among themC. fluminea) on cyanobacterial diets is constrained by a deficiency in essential biochemicals (Martin-Creuzburg et al. 2008;
Basen et al. 2012).
The formation of biodeposition material can be associated with changes in elemental stoichiometry (i.e., the elemental composi- tion of ingested seston can differ from that of biodeposited mate- rial). This has been shown for phosphorus content, which is significantly reduced by bivalve conditioning (Gergs et al. 2009).
Our results indicate similar mechanisms by showing that total fatty acid and PUFA levels are reduced in biodeposited material compared with levels in autotrophs not processed by clams and thus support previous findings obtained with marine bivalves (Bradshaw et al. 1991). However, with regard to cyanobacteria, which did not contain any sterols and long-chain PUFAs, the find- ing that sterols and EPA were detected in cyanobacterial biode- posits suggests that the observed food quality improvement for G. roeseliiwas due to a clam-mediated dietary enrichment with these essential lipids. Although we cannot totally exclude that the small amounts of sterols and PUFAs detected in feces and PSF produced with cyanobacterial diets partially originated from wa- ter microbes passing through the 1m filter in our flow-through system, we propose that these lipids were released by clams dur- ing processing of ingested particles in the mantle cavity (e.g., as part of mucus or excretion products) and then were packed in feces and PSF. This hypothesis is supported byBradshaw et al.
(1991), who already reported an enrichment of individual sterols and fatty acids in biodeposits of a marine bivalve. These sterols and PUFAs were detectable only in feces and PSF produced on cyanobacterial diets (i.e., in the absence of phytoplankton-derived sterols and PUFAs). In feces and PSF produced on eukaryotic algae, these sterols and PUFAs could not be detected, presumably be- cause such small amounts were masked by other sterols and PUFAs originating from the algae in the respective analyses.
In marine amphipods, cholesterol was found to be the dominating sterol (Nelson et al. 2001). Also high levels of PUFAs, especially EPA and DHA, have been reported to occur in gammarid tissues (Nelson et al. 2001;Kolanowski et al. 2007). It was suggested that benthic invertebrates (e.g., gammarids) may selectively consume PUFA-rich particles from sediments to cover their PUFA demands (Goedkoop et al. 2000;Makhutova et al. 2003). In our study, high levels of PUFAs and sterols were provided by the eukaryotic food sources, especially byN. limnetica, which might explain why this alga was of superior food quality forG. roeselii. Additionally, enrichment with PUFAs in S. elongatusfeces (ARA, EPA) andN. limneticaPSF (DHA) may have led to increased growth in juvenile gammarids.
Another important factor determining food quality for aquatic invertebrates is the stoichiometry of carbon, nitrogen, and phos- phorus (Elser et al. 2000;Frost et al. 2002). A stoichiometric mis- match between diet and consumer can lead to lower growth rates of the consumer even at saturating food quantity (Frost and Elser 2002). For example, allochthonous leaves are an important energy source for benthic shredders, but are a low-quality food because of
low phosphorus and nitrogen contents (Cross et al. 2005;Gergs and Rothhaupt 2008). However, in our study elemental stoichiometric constraints can be excluded, because food organisms and their biodeposition material had overall high N and P loads and little to no changes were found in stoichiometry after clam treatment.
Biodeposition constitutes a mechanism that potentially sup- ports benthic food webs. As hypothesized, we found that biode- position by C. fluminea provides a nutritional link between phytoplankton and benthic invertebrates and that this process is associated with an upgrading of phytoplankton food quality. We show that growth and survival of juvenile gammarids on sedi- mented autotrophs differs among autotroph species, but in gen- eral is lower than on the same autotrophs biodeposited by C. fluminea. We also suggest that this upgrading of food quality is partially due to structural changes of food particles during food processing by the clams (i.e., packing in mucus), as direct consumption of sedimented phytoplankton by gammarids is pre- sumably hampered by small cell sizes, but is also due to a clam- mediated supplementation with lipids essential for gammarid growth. In particular, we show that clam conditioning increases the food quality of cyanobacteria forG. roeselii, presumably by providing sterols. Similarly, in experimental pelagic food chains, food quality of cyanobacterial food sources has been shown to be upgraded by heterotrophic protists for subsequent use byDaphnia by dietary provisioning with essential lipids (i.e., sterols and PUFAs;Martin-Creuzburg et al. 2005;Bec et al. 2006). In our exper- iments, we did not provide any additional substrate (sediment or sand) toG. roeseliito be able to clearly relate effects on growth and survival to the provided food sources. It has been recognized, however, that community interactions in sediments (i.e., between bacteria, meiofauna, and macrofauna) can be important for the processing of organic matter (Nascimento et al. 2012). Thus, future work should investigate how benthic–pelagic coupling between bivalves and other benthic invertebrates is affected by benthic community interactions, in particular with regard to a potential channeling of essential nutrients to the detritus-based food web.
Acknowledgements
We thank J. Kim for establishing the setup and helping to con- duct the experiments. This work was funded by the DFG (German Research Foundation) within the collaborative research centre CRC 454 “Littoral of Lake Constance” and the RISE (Research In- ternships in Science and Engineering) program funded by the German Academic Exchange Service (DAAD).
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