Preservation methods alter stable isotope values in gelatinous zooplankton: implications for interpreting trophic ecology

JellyWsh are increasingly topical within studies of marine food webs. Stable isotope analysis represents a valuable technique to unravel the complex trophic role of these long-overlooked species. In other taxa, sample preservation has been shown to alter the isotopic values of species under consideration, potentially leading to misinterpretation of trophic ecology. To identify potential preservation eVects in jellyWsh, we collected Aurelia aurita from Strangford Lough (5422 44.73 N, 532 53.44 W) during May 2009 and processed them using three diVerent methods prior to isotopic analysis (unpreserved, frozen and preserved in ethanol). A distinct preservation eVect was found on N values: furthermore, preservation also inXuenced the positive allometric relationship between individual size and N values. Conversely, C values remained consistent between the three preservation methods, conXicting with previous Wndings for other invertebrate, Wsh and mammalian species. These Wndings have implications for incorporation of jellyWsh into marine food webs and remote sampling regimes where preservation of samples is unavoidable. Introduction Gelatinous zooplankton or jellyWsh (here considered as Phylum Cnidaria, Class Schyphozoa) have been viewed as peripheral and transient components within marine ecosystems, constituting little more than a carbon sink or a trophic dead end (Hansson and Norrman 1995; Arai 2005). This perception now appears outdated and international eVorts are underway to redress this long-standing gap in our knowledge (Mills 2001; Purcell and Arai 2001). However, until recently, many questions surrounding the trophodynamics of jellyWsh seemed somewhat intractable given the spatial and temporal variability of aggregations (Doyle et al. 2007a; Houghton et al. 2007) and the broad scale over which they can occur (Houghton et al. 2006; Doyle et al. 2008). Addressing such bottlenecks is paramount and the recent application of biochemical techniques shows great promise for future studies (see Malej et al. 1993 and Pitt et al. 2009 for review). Advances in stable isotope techniques over the last 20 years have greatly improved ecological research in marine and estuarine systems (Peterson and Fry 1987). The use of nitrogen and carbon stable isotope ratios as food web tracers in marine ecosystems (Carabel et al. 2006; Michener and Kaufman 2007) has made it possible to characterise trophic pathways, aiding the understanding of energy transfer in these systems (e.g. Wada et al. 1987; Kaehler et al. 2000) and allowing us to assess food web structure more accurately (e.g. Peterson and Fry 1987; Hobson and Welch 1992; Davenport and Bax 2002). Biologists seeking to unravel the trophic interactions of jellyWsh face many taxa-speciWc experimental challenges, with three speciWc issues rising to the fore: (1) gut content analysis can be highly problematic as gelatinous prey (e.g. ctenophores) are diYcult to quantify and identify owing to Communicated by U. Sommer. N. E. C. Fleming (&) · J. D. R. Houghton · C. L. Magill · C. Harrod School of Biological Sciences, Medical Biology Centre, Queen’s University, Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK e-mail: [email protected] N. E. C. Fleming · J. D. R. Houghton Queen’s University Marine Laboratory, 12–13 The Strand, Portaferry, Co. Down BT22 1PF, UK 2142 Mar Biol (2011) 158:2141–2146 123 fast deterioration in the oral arms and gut cavity (Båmstedt and Martinussen 2000; Ishii and Tanaka 2001; Pitt et al. 2009); (2) once out of the water, gelatinous samples rapidly lose physical integrity; (3) sampling at remote sites or on research cruises of extended duration can limit access to suitable processing/analytical equipment and jellyWsh collected in the Weld are typically preserved by freezing or storage in ethanol (EtOH) (Hobson et al. 1997). Preservation methods employed prior to stable isotope analysis (SIA) have been shown to alter isotopic composition in a range of marine taxa ranging from algae (Kaehler and Pakhomov 2001; Carabel et al. 2009) through to invertebrates (Bosley and Wainwright 1999; Kaehler and Pakhomov 2001; Carabel et al. 2009) and higher vertebrates (Bosley and Wainwright 1999; Kaehler and Pakhomov 2001). Mateo et al. (2008) additionally highlighted pre-analytical biases at the class level with the greatest eVects exhibited by Maxillopoda, Gastropoda and Polychaeta. Our knowledge of such eVects does not currently extend to gelatinous zooplankton, presenting a potential problem for future studies. Even though the application of SIA to gelatinous zooplankton research is in its infancy, it has the potential to provide a clearer picture of whether each species Wts within established marine food webs or represents a trophic conduit to a separate gelatinous food web. Typically, C is used to investigate energetic pathways through food webs and identify foraging locations used by the individual (DeNiro and Epstein 1978; Wallace et al. 2009). The trophic level at which an organism feeds is reXected by its N value (DeNiro and Epstein 1981; Post 2002). Any mechanism altering the C or N value owing to preservation could lead to incorrect estimation of trophic position and thus overall patterns of consumption, causing problems for further integration into food web and bioenergetic models. SIA provides a useful tool for examining ontogenetic dietary shifts in a range of consumers (e.g. Olson 1996; Harrod et al. 2005; KnoV et al. 2008); however, such patterns have not been examined in jellyWsh. If preservation eVects exist, and are not constant across a gradient of consumer body size, this may aVect our capacity to correctly interpret information regarding trophic ecology from isotope data based on preserved samples. With research on jellyWsh increasing, it is essential to have an a priori knowledge of how diVering preservation methodologies may bias isotopic values. In light of these considerations, the aims of this study were to determine: (1) whether there is an eVect of preservation on jellyWsh stable isotope values, (2) whether SIA could detect any change in isotopic value with increasing body mass (allometry) and (3) if an allometric trend was present, is it aVected by preservation method? In addressing these questions, we sought to establish an eVective pre-analytical processing protocol for jellyWsh. Materials and methods The scyphozoan jellyWsh Aurelia aurita (L.) was selected for the study as it is ubiquitous at temperate latitudes (Russell 1970; Lucas 2001) and has been previously considered in numerous studies of marine food webs (e.g. Lynam et al. 2005; Malej et al. 2007; Purcell et al. 2010). Individuals were collected from a pontoon located at the southern extreme of Strangford Lough (a large coastal embayment Xowing into the Irish Sea) near the Queen’s University Marine Laboratory (5422 44.73 N, 532 53.44 W; Co. Down, Northern Ireland) during May 2009. A dip net (mesh size 1 mm) was used to collect smaller jellyWsh from the side of a small boat, with a landing net (5 mm mesh size) used for larger individuals. Both nets were chosen as they cause minimal damage to jellyWsh tissue (Fleming pers. obs.). To make a direct comparison of the isotopic value of jellyWsh from the same species and same size class, individuals were collected at the same site on the same day but were subsequently randomly assigned to one of three diVerent preservation methods (unpreserved, frozen, EtOH preserved). Originally, we aimed to split each jellyWsh into three sections, and apply each treatment to each individual, allowing comparisons including individual responses to preservation. However, due to the high water content within the bell of jellyWsh (»95%; Doyle et al. 2007b), small individuals did not provide adequate sample mass for replicate samples to be analysed. Our decision was also inXuenced by a wish to mimic procedures on research cruises as closely as possible, where individuals are typically preserved whole. In the laboratory, jellyWsh were rinsed in Wltered seawater, and individual bell diameter (§1 cm) and wet mass (§1 g) recorded. To ensure that preservation treatments were balanced across all size ranges, individuals were sorted by bell diameter in 1 cm increments from 7 to 21 cm. Size-matched samples were then randomly selected and processed following the three diVerent experimental treatments. Unpreserved samples were transferred immediately to drying trays and placed into an oven to attain dry mass (see below for details). Other specimens were either transferred individually into labelled zip-lock bags and frozen at ¡20°C (frozen treatment) or preserved in 75% EtOH and stored at room temperature (EtOH treatment) for 6 months before oven drying using the method described by Doyle et al. (2007b). Frozen and blotted dry EtOH samples were placed into pre-weighed aluminium containers, weighed and placed into a drying oven at a temperature of 60°C and dried to constant mass. Following drying, all samples were ground into a Wne powder using an agate pestle and mortar. The samples were analysed for C and N content and C and N isotope ratios using a Thermo ScientiWc Elemental Analyser Isotope Ratio Mass Spectrometer model: Delta Mar Biol (2011) 158:2141–2146 2143 123 V Advantage. Isotope analysis was carried out at the CHRONO centre, School of Geography, Archaeology and Palaeoecology, Queen’s University, Belfast. Sampling precision for C and N was estimated from the use of internal standards and was typically §0.1‰. Statistical analyses The eVects of preservation were investigated by performing GLM, regression and ANCOVAs on the stable isotope data using SPSS version 17.0. Wet mass and N values were log10-transformed to improve normality, stabilise variances and to linearise relationships.