Stable Carbon Isotope Fractionation in the Marine Copepod Temora Longicornis: Unexpectedly Low Δ13c Value of Faecal Pellets
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MARINE ECOLOGY PROGRESS SERIES Vol. 240: 195–204, 2002 Published September 12 Mar Ecol Prog Ser Stable carbon isotope fractionation in the marine copepod Temora longicornis: unexpectedly low δ13C value of faecal pellets Wim C. M. Klein Breteler*, Kliti Grice**, Stefan Schouten, Hendrikus T. Kloosterhuis, Jaap S. Sinninghe Damsté Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB, Den Burg, The Netherlands ABSTRACT: 13C fractionation effects in an experimental food chain were determined by performing a series of mesocosm experiments with the copepod Temora longicornis and using flagellates as food. 13 The bulk copepods were enriched in C by 1.2 to 2.3‰ and the CO2 respired was enriched by 0.8‰, compared to the isotopic composition of the food. Faecal pellets, however, were strongly depleted in 13C by 4.3 to 11.3‰, in isotopic mass balance with the 13C enrichment of the copepod body and of the respired carbon dioxide. Compound-specific carbon isotope analyses indicated that solvent extractable sterols and alkenones were isotopically light in carbon in both the copepod body and in the faecal matter. These lipids reflected the isotopic nature of the consumed food, indicating that they are not fractionated in the copepod. The residual fraction of the faecal pellets (after extraction) showed the largest isotopic change, being depleted in 13C by about 16‰ compared to the same frac- tion in the diet. Curie point pyrolysis analyses indicated that proteins were the major constituents of this residual material from both the copepods and their faecal pellets. When food sources of different isotopic composition were alternately supplied to the copepods, the faecal pellet residue showed a relatively slow turnover rate compared to the alkenones, which were completely egested by the copepods. A similarly slow turnover rate was observed in the residual material from copepods, sug- gesting that it must be proteins that are being fractionated by the copepod T. longicornis. The low δ13C value of faecal pellets adds another variable to the stable carbon isotopic signature of particulate organic carbon in the pelagic environment. KEY WORDS: Temora · Carbon · Isotope · Fractionation · Faeces · Respiration · Lipid · Protein Resale or republication not permitted without written consent of the publisher INTRODUCTION enriched in 13C relative to their diet, amounting to 0.5 to 1.0‰ per trophic level. Accordingly, isotopic studies Since the early 1980s, 13C/12C isotope ratios of bulk on marine food webs have shown that benthic animals tissue of organisms have been routinely used as tracers and zooplankton are similarly 13C-enriched relative to to determine the pathways of carbon within pelagic the particulate organic carbon (POC) (e.g. Fry & Sherr food webs (see Fry & Sherr 1984 for a review). Previous 1984, Fry 1988). In these studies, the δ13C value of POC work by DeNiro & Epstein (1978) showed that animals is often used as an ‘isotopic reference point’, with the under controlled laboratory conditions are slightly assumption that most POC is derived from phytoplank- ton. However, egesta and body remains of mesozoo- plankton after death contribute a significant propor- **E-mail: [email protected] tion of the POC (e.g. Wiebe et al. 1976, Honjo & Roman **Present address: Centre for Petroleum and Environmental Organic Geochemistry, Department of Applied Chemistry, 1978, Suess 1980, Roy et al. 2000), although actual Curtin University, GPO Box U1987, Perth, Western Aus- amounts vary with location, depth and season (see tralia 6001, Australia review by Corner et al. 1986). © Inter-Research 2002 · www.int-res.com 196 Mar Ecol Prog Ser 240: 195–204, 2002 In the case of copepods, their faecal pellets may We were able to grow algae with a relatively con- partly remain in the pelagic zone, being recycled there stant isotopic composition by using stable continuous by microheterotrophs and also by mesozooplankton cultures with a constant air supply (cf. standard de- (coprophagy) (Urban-Rich et al. 1999). Depending on viations in Table 1). For the heterotrophic Oxyrrhis diet, pellet size, turbulence regime and water depth, marina we assumed a δ13C value of –13.3 ± 0.4‰ a significant proportion may rapidly sediment and (mean ± SD, n = 5) observed in a continuous culture by thereby supply the deep sea with comparatively fresh van Rooy (1989). The isotopic composition of the mixed organic matter (Mauchline 1998, Urban-Rich 2001). diet used to feed the copepod stock culture was calcu- Therefore, when carbon isotopic signatures are deter- lated as the average value of O. marina and the 2 food mined in food web studies, it is important to know the species present in this mixture (see Table 1). contribution of faecal pellets to the δ13C value of POC, Feeding experiments. To determine isotopic frac- which may largely depend on the degree of fractiona- tionation of the consumed food, nearly mature Temora tion during digestion and assimilation in the grazers. In longicornis (copepodite stages CIV, V, and adults) the present research we address the following specific were adapted for a period of 4 d to a single food source questions: (1) How do the carbon isotopic values of of constant isotopic composition, during which they algae, copepods and faecal pellets compare? (2) What largely matured. The copepods were transferred to 22 l are the major biochemical components of faecal pel- of filtered (2 µm) seawater and fed with Isochrysis lets? (3) Is there a different assimilation, fractionation galbana or Rhodomonas sp. at a concentration of and turnover rate of different food components? (4) Is it ~300 µg C l–1. A nylon mesh with 200 µm apertures was possible to establish an isotopic mass balance? To the suspended in the 22 l tank about 2 cm from the bottom, best of our knowledge, this is the first attempt to estab- to separate the faecal pellets from the copepods. Every lish an isotopic mass balance for an aquatic organism. other day, the copepods, a quarter of the seawater (and We conducted a series of controlled laboratory ex- algal food), and the 200 µm mesh were transferred into periments with cultured marine algae and copepods. a new tank of 2 µm filtered seawater. Several hundreds The algal food and the copepods, eggs, exuviae, faecal of faecal pellets were collected, typically every 2 d 13 pellets and respired CO2 were analysed for their C under dim light, by sequential filtration (200, 50 and composition. The 13C composition of the residual mat- 20 µm mesh), washed with double-distilled water and ter (after extraction) of some of these materials was centrifuged to remove any floating debris. The remain- determined and compared to that of solvent-extracted ing debris was removed with a Pasteur pipette under a lipids measured by Grice et al. (1998) in related work. dissecting microscope. All samples of faecal pellets The main biochemical classes in the faecal pellets and were frozen to minimise bacterial degradation (Honjo exuviae were determined in comparison to those of the & Roman 1978), and combined to obtain sufficient algae and the copepods. material for analysis. The experiments were carried out in the dark at 15°C for a period of up to 1 wk. At the end of the experiment, most of the copepod bodies MATERIALS AND METHODS and an aliquot of the algae from the overflow of the continuous cultures were also collected, frozen and Culture conditions. Broodstock of the copepod retained for analyses. Temora longicornis (Müller) was continuously cultured In one of the experiments with Isochrysis galbana in the laboratory at 15°C. The cryptophyte Rhodo- as food, the δ13C value of the food was altered, using 2 monas sp. (4.8 to 8.5 µm equivalent spherical diameter, I. galbana cultures with different isotopic compositions, ESD) and the haptophyte Isochrysis galbana (3.6 to to trace the flow of carbon through the experimental 7.0 µm ESD), both grown non-axenically in chemo- food chain. One algal culture was supplied only with stats, were supplied as food in surplus of the copepods’ air, while the other was supplied with a mixture of air –1 needs (>300 µg C l ). The heterotrophic dinoflagellate and isotopically light CO2. The isotopic composition of Oxyrrhis marina (10.4 to 19.0 µm ESD) was grown in the second culture was allowed to stabilise for a period a separate 2-stage continuous culture using Rhodo- of 2 wk prior to the experiment. Temora longicornis monas sp. as a single food source. O. marina also were fed with the first (standard) culture of I. galbana occurred in the copepod stock cultures, competing during 4 d of adaptation and during the following first with the copepods for the same food algae, but at the 4d of the experiment. Thereafter, the carbon isotopi- same time O. marina was an important food source for cally light I. galbana culture was fed to the copepods for the copepods. In the experiments with single species of 6 d, after which the food was switched back to the first algae as food, O. marina was carefully removed. For I. galbana culture for a further 4 d. Collection and treat- further details on culture conditions, see Klein Breteler ment of samples of algae, copepods and faecal pellets & Gonzalez (1986, 1988). were the same as in the other feeding experiments. Klein Breteler et al.: Carbon isotopic fractionation in copepods 197 Exuviae and eggs of copepods were collected from which contained a 12 mm combusted Whatman GF/C the copepod broodstock, using a dissecting micro- filter soaked with 0.05 ml 10M NaOH (Schuster et al. scope, and analysed similarly to the other materials. 1998). The CO2 was collected on the filter over a 24 h Lipids were extracted from part of the material and period.