Experimental study on the effect of diet on fatty acid and stable isotope profiles of the squid Lolliguncula brevis

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1 Journal of Experimental Marine Biology and Ecology 333 (26) Experimental study on the effect of diet on fatty acid and stable isotope profiles of the squid Lolliguncula brevis Gabriele Stowasser a,b,, Graham J. Pierce a, Colin F. Moffat c, Martin A. Collins b, John W. Forsythe d a School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK b British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 ET, UK c FRS Marine Laboratory, 375 Victoria Road, Aberdeen, AB11 9DB, UK d National Resource Center for Cephalopods, Marine Biomedical Institute, University of Texas Medical Branch, 31 University Boulevard, Galveston, Texas, TX , USA Received 29 June 25; received in revised form 18 August 25; accepted December 25 Abstract Fatty acid and stable isotope analyses have previously been used to investigate foraging patterns of fish, birds, marine mammals and most recently cephalopod species. To evaluate the application of these methods for dietary studies in squid, it is important to understand the degree to which fatty acid and stable isotope signatures of prey species are reflected in the squids' tissue. Four groups of Lolliguncula brevis were fed on prey species with distinctly different fatty acid and stable isotope profiles over 3 consecutive days. One group of squid were fed fish for fifteen days, followed by crustaceans for a further fifteen days. A second and third group were fed exclusively on fish or crustaceans for thirty days. And a fourth group was fed on a mixture of fish and crustaceans for thirty days. Analysis of squid tissue showed that, after days of feeding, fatty acid profiles of squid tended to reflect those of their prey. Squid that fed on a single prey type, i.e. fish or crustacean, showed only minor modifications in fatty acid proportions after the initial change and fatty acid profiles were clearly distinguishable between the two feeding groups. Shifts in fatty acid proportions towards respective prey profiles could clearly be observed in squid the diet of which was swapped after 15 days. Clear differences could also be seen in fatty acid profiles of squid feeding on a mixed diet with trends towards either fish or crustacean fatty acid signatures. Stable isotope signatures of squid tissues clearly distinguished between animals feeding on different diets and supported findings from fatty acid analysis, thus indicating both methods to be viable tools in feeding studies on squid species. 26 Elsevier B.V. All rights reserved. Keywords: Cephalopods; Fatty acids; Feeding experiment; Lolliguncula brevis; Stable isotopes 1. Introduction Corresponding author. British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 ET, UK. Tel.: ; fax: address: gsto@bas.ac.uk (G. Stowasser). Cephalopods are active predators at all stages of their life and are generally regarded as opportunistic, taking a wide variety of prey (Rodhouse and Nigmatullin, 1996). They also sustain a wide variety of marine top predators such as fish, birds and marine mammals (e.g. Croxall /$ - see front matter 26 Elsevier B.V. All rights reserved. doi:.16/j.jembe

2 98 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) and Prince, 1996; Smale, 1996; Santos et al., 21). Due to the scale of their biomass (estimated worldwide standing stock of Mt, Rodhouse and Nigmatullin, 1996), cephalopods are believed to have a significant impact on species at higher and lower trophic levels. Evidence exists in several areas that fishing pressure has changed ecological conditions and that shifts in community structure have occurred, with cephalopod stocks partially replacing predatory fish stocks (e.g. Caddy and Rodhouse, 1998). As cephalopods are increasingly targeted by fisheries, it is necessary to develop management strategies that ensure the sustainability of populations and provide tools for controlling fishing effort. In order to develop efficient management strategies for squid species a detailed knowledge of their trophic relationships is required, as predation has a significant influence on community structure and population dynamics (Paine, 1988). Although numerous feeding studies have been carried out, the importance of many cephalopod species as predators is still not well known, reflecting the difficulty of obtaining data and biases in data available from stomach contents analysis. In cephalopods, since the oesophagus passes through the brain, food is broken up into small pieces prior to ingestion and some hard parts may not be ingested (see Rodhouse and Nigmatullin, 1996). In recent years, more sophisticated methods for dietary analysis have been developed. Fatty acid and stable isotope analyses were found to be valuable tools in examining trophic relationships and the transport of organic matter along food chains (e.g. Sargent, 1976; Fry, 1988). These techniques offer advantages over the use of stomach contents analysis as they can be applied to any animal, even if the stomach is empty, they provide information on average diet, integrated over a period of time, and have the potential to allow improved quantitative analysis (Iverson et al., 24). Specific fatty acids are known to be characteristic of certain marine species and can be used as dietary markers in feeding studies (Ackman et al., 197; Sargent et al., 1987). These so-called biomarkers consist of fatty acids (e.g. the polyunsaturates [PUFAs] 18:2 n-6 and 22:6 n-3) that cannot be biosynthesised by the study animal but have to be taken up with the diet. Fatty acid content can also provide information about origin of the diet (i.e. the feeding habitat) as, for example, ratios of certain PUFAs (e.g. of the n-3 family to the n-6 family) are significantly lower in freshwater than in marine environments (Sargent, 1976). As with fatty acids, the utility of stable isotopes arises from the fact that isotope abundances in a predator are strongly influenced by the isotopic abundances of its prey (Fry and Sherr, 1989). δ 15 Nitrogen levels (i.e. a measure of the prevalence of atoms of 15 N in relation to that of 14 N) show an average stepwise enrichment of 3 5 with each change in trophic level (Hobson, 199). Differences in δ 15 Nitrogen levels between species therefore allow us to assess a species' position in the food web (e.g., Peterson, 1999). δ 13 Carbon levels on the other hand increase on average by only 1 at every step in the food chain and indicate a predator's distance from the food base in its trophic system (Michener and Schell, 1994). In most studies, δ 13 Carbon analysis is used to identify the habitat of a species, by discriminating between carbon levels of different ecosystems, e.g. freshwater vs. marine, pelagic vs. benthic and offshore vs. inshore, and latitudinal differences (e.g. Rau et al., 1982; Smith et al., 1996). The stable isotope and fatty acid compositions of different tissues within the same organism reflect different periods in dietary history, as each tissue has a different metabolic rate and, consequently a different turnover rate. This will allow, in theory, an evaluation of diet integrated over different periods of time according to the tissues studied (e.g. Hobson et al., 1996). Only in recent years has fatty acid analysis been applied to study the trophic ecology of cephalopod species (e.g. Phillips et al., 22; Navarro and Villanueva, 23). Stable isotopes of cephalopods have been analysed mainly in the context of these species being prey to higher trophic levels (e.g. Hobson et al., 1994) and only two studies investigate the role of cephalopod species as predators (Takai et al., 2; Cherel and Hobson, 25). No previous feeding study uses the combined approach of fatty acid and stable isotope analysis to determine diet in cephalopod species. The brief squid Lolliguncula brevis (Blainville, 1823) is a common inshore squid species of the Gulf of Mexico and western Atlantic coast (Vecchione, 1982). It displays high growth rates and shows lower susceptibility to fin damage and intraspecific aggression in captivity than most other squid species taken from the wild (Hanlon et al., 1983). This hardiness and its yearround availability make it a potentially useful species for long-term in vivo experiments (Hanlon et al., 1983) and justify its selection as the experimental species for this study. The present feeding study was designed to evaluate the impact of diet on the fatty acid and stable isotope signatures in different tissues of L. brevis and thus

3 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) evaluate the utility of these methods in investigating the trophic ecology of cephalopod species. 2. Material and methods 2.1. Animal maintenance and tank system This study was carried out at the National Resource Center for Cephalopods (NRCC) in Galveston, Texas, USA, where L. brevis is routinely maintained in captivity. Sixty-five specimens of L. brevis were caught in May 21 in the Galveston shipping channel and Galveston Bay (Northern Jetty) by beach seining and bottom trawl, respectively. Animals were transferred to a movable acclimatisation tank, which was filled with seawater at the capture location. At the laboratory, the acclimatisation tank was connected to an aquarium with recirculating natural seawater, conditions being kept as close as possible to the squids' natural habitat. Five animals were sacrificed immediately after capture to provide the baseline for both the fatty acid and stable isotope signatures. After an acclimatisation period of 48 h, the remaining 6 squid were transferred to the experimental aquarium. Squid were then left to adjust to aquarium conditions for a further 24 h. Animals were not fed prior to the start of the experiment. The flow-through experimental tank ( m) was sectioned off by solid partitions into five similar-sized sections of approximately 1.15 m length. The first four sections held the squid and the last section contained the pump and filter system. The water used was natural seawater collected from the Galveston Bay area. Water depth ranged from 4 45 cm over the course of the experiment. Water temperature fluctuated between 19 and 21 C with a mean salinity of 26 ppt. Temperature and salinity were recorded on a daily basis. To reduce water turbidity a filter system of polyester fibre and activated carbon in the filter bed assured the separation of particulate organic carbon and adsorption of dissolved organic carbon, respectively. To monitor water quality, ph and nitrate levels were measured weekly. To keep levels at an optimum (ph ; nitrate below 5 ppm, Walsh et al., 22) filter fibre and activated carbon were changed twice over the course of the experiment and food remains were siphoned out of the tanks on a daily basis. Fifteen squid were initially placed into each of the four sections. In order to reduce intersexual aggression, an effort was made to separate females and males, based on external morphology Food and feeding regime Species given as food during this experiment are part of the natural diet of L. brevis (Hanlon et al., 1983 and Table 1). The majority of prey was caught by seining every 2 3 days in estuaries and marshes along the coastline of Galveston Island. To be able to compare a wider range of prey profiles to unmodified squid profiles, an additional eight putative prey species were collected in Galveston Bay (Northern Jetty, Table 1). Squid were fed twice daily over 3 consecutive days. Squid were initially fed on 2 3 prey items per feed over the first two days. In all sections, some prey animals remained uneaten and tended to form aggregations, thus avoiding predation. Consequently remaining live prey was netted out of the tank and feeding was reduced to 1 2 prey items per squid per feed for the remainder of the experiment. The capture and feeding of live prey was carried out under permit (Scientific Permit Number SPR-59-69, under the authority of Chapter 43, Table 1 Putative prey species collected for feeding (Estuaries) and biochemical analyses (Estuaries and Northern Jetty) Species Common name Area Samples [n] Analyses Pisces Menidia beryllina Silverside Estuaries 6 FA (16); SI (4) Cyprinodon variegatus Sheepshead minnow Estuaries 45 FA (13); SI (3) Poecilia latipinna Sailfin molly Estuaries 4 FA (12); SI (3) Mugil cephalus Striped Estuaries 3 FA (3) mullet Micropogonias undulatus Atlantic croaker Northern Jetty 2 FA (1); SI (2) Cynoscion arenarius Sand trout Northern Jetty 2 FA (2); SI (1) Menticirrhus Southern Northern 1 FA (1); americanus kingfish Jetty Polydactylus Atlantic Northern 1 FA (1) octonemus threadfin Jetty Brevoortia patronus Gulf menhaden Northern Jetty 1 FA (1); SI (1) Dorosoma petenense Threadfin shad Northern Jetty 5 FA (5) Menidia beryllina Crustacea Palaemonetes pugio Penaeidae spp. Silverside Northern Jetty 3 FA (3); Grass shrimp Estuaries 76 FA (16); SI (5) Pinaeid Northern 3 FA (3); shrimp Jetty SI (1) FA=fatty acid analysis; SI=stable isotope analysis. Numbers in brackets indicate numbers examined per type of analysis.

4 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) subchapter C of the Texas Parks and Wildlife Code) issued to the NRCC, Galveston. Squid in each feeding section of the tank (I IV) were given a different diet, as follows: Feeding group I fed exclusively on fish for 15 consecutive days (SF, i.e. switched diet, fish), then crustaceans for 15 consecutive days (SC, i.e. switched diet, crustacean). Squid were sacrificed after 15 and 3 days. Feeding group II fed exclusively on fish over the whole feeding period (F, all fish diet). Samples were taken after, 2 and 3 days. Feeding group III fed exclusively on crustaceans over the whole feeding period (C, all crustacean diet). Samples were taken after, 2 and 3 days. Feeding group IV fed on a mixed diet of crustaceans and fish over the whole feeding period (Mixed). Samples were taken after 3 days. For the remainder of the text, feeding groups will be referred to by their group acronyms, i.e. SF, SC, F, C and Mixed. The group wild refers to the 5 animals that were sacrificed prior to the start of the feeding experiment. Concerning prey, bay species refers to fish and crustaceans caught at the Northern Jetty in more saline waters. Estuarine species refers to fish and crustaceans caught in brackish waters from estuaries and marshes around the island and which were used for feeding Sample analyses Three to five squid were sacrificed 1 h after the first feed of the day, on sampling days. Numbers of squid taken per sampling event were adjusted to take account of any natural mortality occurring in each section so that an equal number of animals remained in each section after each sampling event. Two tissues, digestive gland and muscle, were chosen to determine diet changes within the same organism as these are hypothesised to integrate diet over different periods of time. The digestive gland data are expected to reflect very recent feeding whereas muscle data will reflect diet over a longer period of time. Reference groups of both squid and prey species (other than those used in the experiment) were taken to investigate feeding in the wild and for comparison with animals used in the experiment. Basic biological data, i.e. dorsal mantle length, sex and maturity (maturity index, Hixon, 198) were recorded and tissue samples were taken. Animals that died of natural causes were processed in the same way; these animals would have been sampled within a maximum of 12 h of death. Tissues were stored frozen at 8 C prior to biochemical analyses. Prey samples were collected on each day of capture and stored whole ( 8 C) until further analysis. Fatty acid analysis was carried out on 58 squid used in the experiment as well as the five wild specimens. For prey species of which high numbers of individuals were collected (e.g. silverside, sheepshead minnow, sailfin molly and the grass shrimp), after the initial examination of single specimens, analysis was carried out on batches of 3 5 animals. Lipids were isolated from samples of the squid digestive gland and muscle tissue, while extractions were made from whole animals in the case of fish and crustacean prey species. Total lipid was extracted using a chloroform methanol water solvent (2: 2: 1.4 v/v/v) mixture, following the standard operating procedure (SOP) for lipid analysis at FRS Marine Laboratory, Aberdeen (after Bligh and Dyer, 1959; Hanson and Olley, 1963). Total lipids were transesterified overnight (incubated at 5 C) using methanol containing sulphuric acid (1% v/v). The resulting methyl esters were extracted into isohexane and stored over anhydrous sodium sulphate at 2 C until further analysis. Fatty acid methyl esters (FAME) were analysed by gas chromatography with flame ionisation detection (GC-FID) on a Hewlett Packard 589 Series II gas chromatograph, fitted with a fused silica capillary column (.25 mm i.d. 3 m length) coated with a polar DB-23 phase (J and W Scientific Inc., California, USA), using nitrogen as the carrier gas. The detector temperature was set at 3 C. To achieve optimum separation of components the column temperature was ramped from 6 C initial oven temperature at 25 C min 1 to 15 C, then ramped at 1 C min 1 up to 2 C and held for min, then ramped at C min 1 to 22 C and finally held at 22 C for 5 min. Oncolumn injection (1 μl) was by means of a Hewlett Packard 7673 automatic injector. Twenty-five fatty acids (Tables 2a,b) were identified through reference to a standard of known composition, and comprise the fatty acid population chosen for this assessment. Amounts present are expressed as percentages of the total area for these fatty acids (NA, normalised area percentage). The areas for the isomers of the monounsaturated fatty acids 18:1 (n-9 and n-7) and 2:1 (n-11 and n-9) were combined and the combined area is signified by 18:1 and 2:1, respectively.

5 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) Table 2a Mean fatty acid signatures in digestive gland tissue of L. brevis Species L. brevis Wild L. brevis SF L. brevis SC L. brevis F L. brevis C L. brevis Mixed N samples Total lipid (%).19± ± ± ± ± ± :.9±.4.±.9.9±.3.6±.4.±.4.17±.33 14: 1.57± ± ± ± ± ±.9 15:.54±.9.74±.31.53±.5.59±.17.53±..65±.42 16: 19.63± ± ± ± ± ± :1 n-7 2.± ± ± ± ±.99 3.± :2 n-6.5±.7.19±.18.21±.14.16±.15.7±.9.18±.18 16:3 n-6.15±.13.32±.23.42±.48.37±.34.2±.32.42±.41 16:4 n-3.14±.12.3±.3.1±.2.8±.11.6±.6.5±.6 18: 11.5± ± ± ±2.2.53± ± :1 n-9 3.8± ± ± ± ± ± :1 n ± ± ± ± ± ±.98 18:2 n-6.62± ± ± ± ± ±.94 18:3 n-6.29±.3.37±.17.27±.6.36±.18.26±.14.31±.15 18:3 n-3.46±.29.62±.41.7±.39.52±.36.88±.4.6±.37 18:4 n-3.23±.11.9±.8.4±.4.11±.7.8±.8.7±.5 2:1 n ± ± ± ± ± ±.56 2:1 n-9.3±.7.3±.7.25±.16.32±..8±.82.53±.48 2:4 n ± ± ± ± ± ±3.37 2:4 n-3.28±.5.34±.14.2±.3.25±.9.21±.7.23±.11 2:5 n ± ± ± ± ± ± :1 n-11.46±.43.15±.4.14±.5.15±.8.±.9.22±.2 21:5 n-3.5±.65.±.7.21±..16±.16.21±.12.12±.7 22:5 n ± ± ± ± ± ±.54 22:6 n ± ± ± ± ± ± :1 n-9.83±.6.25±.15.8±.3.27±.18.11±.12.18±.16 SAT 33.32± ± ± ± ± ±2.88 MUFA 11.34± ± ± ± ± ±3.14 PUFA 55.34± ± ± ± ± ±3.97 n-3/ n-6 ratio 8.89± ± ± ± ± ±1.51 Fatty acids are given in normalised area percentage () ±standard deviation of all fatty acids determined. indicates fatty acids selected for statistical analysis. SAT = saturated fatty acids, MUFA=monounsaturated fatty acids, PUFA=polyunsaturated fatty acids. Feeding sections: SF=switched diet/fish (day 1 15), SC = switched diet/crustacea (day 16 3), F=all fish (3 days), C = all crustacea (3 days), Mixed=fish and crustacea (3 days). Stable isotopes were analysed from digestive gland and muscle tissue of 29 squid, taken from the start, middle and end of the experiment. Stable isotope analysis of prey was carried out on several individual specimens of selected representative species only (Table 1). Tissue samples were freeze-dried (Edwards Super Modulyo freeze dryer, Edwards Ltd., UK). Dried samples were powdered with a mortar and pestle and approximately 1 mg loaded into a 8 4 mm tin capsule (Europa Scientific Ltd, Crewe, UK). Duplicate samples were combusted in a Carlo Erba Na 15 NC Elemental Analyser at 18 C flash combustion temperature. Resulting CO 2 (g) and N 2 (g) were analysed for carbon and nitrogen stable isotope ratios, respectively, with a dual inlet mass spectrometer (VG Micromass OPTIMA, VG Ltd., Manchester, UK). Internal laboratory standards were run at regular intervals. Stable-isotope concentrations were expressed in δ notation as parts per thousand ( ) difference from a standard reference material calculated according to the following equation: dx ¼½ðR sample =R standard Þ 1Š where X is 13 Cor 15 N, R is the corresponding ratio 13 C/ 12 Cor 15 N/ 14 N, and δ is the measure of the ratio of heavy to light isotope in the sample. Values measured were raw mass spectrometry δ estimates relative to laboratory working standards and had to be adjusted to international standards. δ estimates were adjusted to international standards IAEA CH-6, NBS-19 and IAEA CO-1 (calibrated against carbon standard material Peedee belimnite (PDB)) and international standard IAEA 35 N (calibrated against atmospheric N 2 (AIR)), respectively. Analytical error was.4 to.7 and

6 2 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) Table 2b Mean fatty acid signatures in muscle tissue of L. brevis Species L. brevis Wild L. brevis SF L. brevis SC L. brevis F L. brevis C L. brevis Mixed N samples Total lipid (%) 3.37± ± ± ± ± ±.83 12:.12±.5.8±.4.7±.2.5±.2.±.4.6±.3 14: 3.35± ± ± ± ± ±.69 15:.62±.7.65±.8.62±.7.54±.9.55±.4.55±.9 16: 24.13± ± ± ± ± ± :1 n-7 1.± ± ± ± ± ±.32 16:2 n-6.4±.9.2±.2.4±.5.2±.3.9±.11.±.8 16:3 n-6.23±.8.33±.7.28±.24.4±..42±.4.45±.31 16:4 n-3.3±.7.2±.2.±.11.2±.2.1±.1.5±.7 18: 7.78± ± ± ± ± ±.67 18:1 n ± ± ± ± ± ±1.1 18:1 n ± ± ± ± ± ±.49 18:2 n-6.3±.5.65± ±.66.5± ±.45.92±.4 18:3 n-6.3±.4.22±.11.18±.9.28±.7.23±.12.2±.7 18:3 n-3.17±..22±.6.37±.17.17±..48±..31±.15 18:4 n-3.16±.4.7±.3.5±.2.6±.3.5±.4.6±.3 2:1 n ± ± ± ± ± ±.24 2:1 n-9.51±.15.29±.29.32±.27.2±.8.61±.63.2±.12 2:4 n ± ± ± ± ± ±1.82 2:4 n-3.3±.12.26±.6.25±.4.2±.3.24±.6.2±.4 2:5 n ± ± ± ± ± ±1.9 22:1 n-11.47±.7.32±.8.23±.16.35±.8.3±.12.28±.11 21:5 n-3.13±.7.14±.2.13±.2.13±.4.33±.69.13±.4 22:5 n-3 1.9± ± ± ± ± ±.54 22:6 n ± ± ± ± ± ± :1 n-9.37±.11.19±.2.15±.9.17±.16.26±.5.7±. SAT 36.± ± ± ± ± ±1.7 MUFA.44±.93.62± ±1.28.2± ± ±1.37 PUFA 53.57± ± ± ± ± ±1.54 n-3/ n-6 ratio 14.8± ± ± ± ± ±3.99 Fatty acids are given in normalised area percentage () ±standard deviation, of all fatty acids determined. indicates fatty acids selected for statistical analysis. SAT=saturated fatty acids, MUFA=monounsaturated fatty acids, PUFA=polyunsaturated fatty acids. Feeding sections: SF=switched diet/fish (day 1 15), SC=switched diet/crustacea (day 16 3), F=all fish (3 days), C=all crustacea (3 days), Mixed=fish and crustacea (3 days)..3 to.7 for carbon and nitrogen, respectively, depending on standards used Statistical analysis Of the 25 fatty acids chosen, 18 of the most abundant and dietary relevant fatty acids in squid and/ or prey species were selected for statistical analysis (as indicated by in Tables 2a,b). In all further analyses these were classed as major and minor fatty acids, based on their level of contribution to the fatty acid profile, with minor fatty acids being b5% (single fatty acids). Between-section differences in selected fatty acids as well as differences in fatty acid composition between squid dying of natural causes and squid sacrificed were examined using one-way ANOVA on arcsine-transformed data. Balanced multivariate analysis of variance (MAN- OVA) on arcsine-transformed fatty acid data was applied to test for differences between sections. Due to unequal sample sizes, results on animals that were subjected to a diet swap (SF and SC) had to be excluded from this analysis and one randomly selected sample from F and Mixed each were removed for this multivariate analysis. Principal component analysis (PCA) on arcsine-transformed fatty acid and stable isotope data was used to look for groupings in data according to feeding type and correlations between predator and prey signatures. 3. Results The number of females used in the experiment (n=5) exceeded that of males (n=13). Female dorsal

7 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) mantle lengths (DML, range mm) were on average larger than male DML (range 4 63 mm). Maturity stages ranged from 1 to 4 in female squid, with 59% of animals belonging to maturity stage 4 (i.e. fully mature). In male squid maturity stages ranged from 2 to 5 with 36% of animals being fully mature Fatty acid analysis Potential prey Prey species used for feeding experiments varied significantly in proportions of all fatty acids (ANOVA, p b.1 for all FA). This interspecific variation was mainly due to the large differences in fatty acid ratios between grass shrimp and the fish species. Fatty acid signatures for grass shrimp showed comparatively high levels of 18:1, 18:2 n-6 and 2:5 n-3 and very small proportions of 22:5 n-3 and 22:6 n-3. Fish, on the other hand, showed higher levels of 18:, 2:4 n-6, 22:5 n-3 and 22:6 n-3. Prey species collected from bay waters showed higher values for the C2-and C22-based PUFAs whereas estuarine species showed higher proportions of monounsaturates and C18 PUFAs (Table 3). Furthermore, n-3/ n-6 ratios (range ) were on average higher in bay species than in species collected from estuarine waters (range ). Differences were mainly due to higher proportions of PUFAs of the n-6 series such as 18:2 n-6 and 2:4 n-6 in estuarine species and correspondingly lower proportions of n-3 PUFAs 2:5 n-3 and 22:6 n-3. High within-species variation was found in silverside and sheepshead minnow. The three specimens of silverside caught in the bay showed significantly lower proportions of 18:1, 18 PUFA, and 2:4 n-6, and higher proportions of 2:5 n-3 and 22:6 n-3, than Table 3 Mean fatty acid signatures in prey species used in the experiment Species Silverside Sailfin molly Sheepshead minnow Striped mullet Grass shrimp N samples Total lipid (%) 2.27± ± ± ± ±.66 12:.14±.6.17±.5.±.5.6±.2.18±.1 14: 1.95± ± ±1.3 1.± ±.12 15:.87±.21 2.± ± ± ±.5 16: 2.48± ± ± ± ±.37 16:1 n ± ± ± ± ±.8 16:2 n-6.17±.8.41±.22.44±.26.19±.9.25±.3 16:3 n-6.35±.42.68±.52.47±.27 3.± ±.73 16:4 n-3.19±.15.±.5.21±.13.8±.6.5±.3 18: 7.73± ± ± ± ±.37 18:1 n-9.63± ±3.25.6± ± ±.74 18:1 n ± ± ± ± ±.46 18:2 n ± ± ±1.5.97± ±.74 18:3 n-6 1.± ±.43.8±.26.74±.28.74±.8 18:3 n ± ± ±.59.26± ±.33 18:4 n-3.49±.21.48±.2.46±.3.22±.4.22±.6 2:1 n-11.44±.13.59±.27.56± ±.42.36±.6 2:1 n-9.58±.51.36±.27.62±.16.31±.14.3±.4 2:4 n ± ± ± ± ±.76 2:4 n-3.7±.32.39±.7.58±.2.35±.3.25±.4 2:5 n ± ± ± ± ±.73 22:1 n-11.14±.8.21±.13.2±.11.34±.4.8±.4 21:5 n-3.16±.6.14±.5.32±..27±.5.9±.2 22:5 n ± ± ± ±1.3.65±. 22:6 n ± ± ± ± ±.86 24:1 n-9.61±.32.16±.6.29±.19.33±.15.5±.2 SAT 31.11± ± ± ± ±.5 MUFA 21.76± ± ± ± ±1.59 PUFA 47.13± ± ± ± ±1.64 n-3/ n-6 ratio 3.91± ± ± ± ±.22 Fatty acids are given in normalised area percentage () ±standard deviation of all fatty acids determined. SAT=saturated fatty acids, MUFA=monounsaturated fatty acids, PUFA=polyunsaturated fatty acids.

8 4 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) the conspecifics caught in estuarine waters. Consequently silversides from bay waters had n-3/ n-6 ratios 2 4 times higher than silversides from estuarine waters. Their fatty acid signatures were more similar to those of other bay species than to those of their conspecifics Wild Lolliguncula brevis The array of fatty acids identified was the same for both muscle and digestive gland in L. brevis (Tables 2a,b). Proportions of individual fatty acids however differed significantly between tissue types. Digestive gland contained significantly higher amounts of 18:, 16:1 n-7, 2:4 n-6 and 2:5 n-3 than muscle, and significantly lower amounts of 14:, 16: and 22:6 n- 3. With the exception of 18:, intraspecific variation in fatty acids was always higher in digestive gland tissue. Although squid fatty acid signatures could mostly be differentiated from those of estuarine and bay prey species, some similarities in proportions of individual fatty acids were seen. Low proportions of 16:1 n-7, C 18 PUFA and high proportions of 2:5 n-3 and 22:6 n-3 were frequently found in squid tissue (digestive gland and muscle) and in bay fish species. Average values of n-3/n-6 ratios of squid were closer to marine rather than estuarine prey species Feeding experiment To test for differences in fatty acid signatures between squid found deceased in the feeding tank and squid sacrificed 1 h after feeding, results for single fatty acids in both groups were compared, within each feeding section and for each tissue. Although significant differences between the two categories of squid were found for individual fatty acids, within each category differences between feeding groups were similar. Thus, over time, fatty acid profiles changed towards prey fatty acid signatures in squid of both categories for both tissues. Therefore, although there were differences between squid that were sacrificed and those dying of natural causes, data from all squid were treated together during subsequent analyses except where otherwise stated Differences in fatty acid composition between feeding groups (F, C and Mixed). The fatty acid composition of digestive gland tissue varied considerably between squid fed on different diets (MANOVA, pb.1, Fig. 1a, b). Tissues of squid fed on fish (F) showed higher levels of 18:, 2:4 n-6 and 22:6 n-3 compared to animals fed exclusively on crustaceans (C). In contrast, the latter showed higher levels of 18:1, 18:2 n-6 and 2:5 n-3. Squid fed on a mixed diet showed proportions of fatty acids intermediate between those found for groups F and C. In the case of fatty acids for which no significant variation was found between sections, proportions nevertheless broadly resembled those for prey given (Fig. 1 a d). Ratios of n-3 PUFA to n-6 PUFA changed within the first few days of the experiment. The highest ratios were found for animals sacrificed at the beginning of the experiment but ratios dropped to the same average level in all three sections after ten days into the experiment (Table 2a). Muscle tissue produced similar results to digestive gland tissue, with significant variation in fatty acid composition between feeding groups (MANOVA, pb.1). Due to low variation within feeding groups for single fatty acids in muscle, more fatty acids turned out to vary significantly between feeding groups. Fatty acid proportions in muscle tissue of squid fed on a mixed diet were intermediate between those found for fish and crustacean diets. As in digestive gland tissue, the ratios of n-3/n-6 dropped from the start of the experiment to a rather constant level within a short period (Table 2b). Squid fed on crustaceans showed slightly lower ratios than squid fed on fish, although differences between sections were not significant. Though still distinguishable from their prey, after 4 weeks of feeding, the ratios of fatty acids in squid more closely resembled those of their prey Temporal changes in fatty acid composition. Tissues of squid in groups SF (fish diet) and SC (crustacean diet) displayed clear differences in fatty acid composition after the switch in diet from fish to crustacean food. Pooled over 15 days of feeding, digestive gland tissue of squid fed on crustaceans showed higher proportions of 18:1, 18:2 n-6, 18:3 n- 3 and 2:5 n-3, and lower proportions of 2:4 n-6, 22:5 n-3 and 22:6 n-3, compared to squid fed on fish (Fig. 2). Muscle tissue of squid fed on crustaceans showed higher levels of 18:1, 18:2 n-6, 18:3 n-3, 2:4 n-6 and 2:5 n-3, and lower levels in 22:6 n-3, than muscle of squid fed on fish. In digestive gland tissue, average ratios of n-3/ n-6 were the same for both feeding regimes, while ratios in muscle tissue were lower for squid fed on a crustacean diet than for squid fed on fish. The most pronounced differences in fatty acid profiles were found between squid sacrificed at the start of the experiment and squid sacrificed after days

9 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) a.) Fish diet Crustacean diet Mixed diet b.) : 16:1n-7 18: 18:1* 18:2n-6 18:3n-3 2:4n-6 2:5n-3 22:5n-3 22:6n-3 c.) 3 Silverside Minnow Grass shrimp Sailfin molly Striped mullet d.) : 16:1n-7 18: 18:1* 18:2n-6 18:3n-3 2:4n-6 2:5n-3 22:5n-3 22:6n-3 Fig. 1. (a) and (b) Selected fatty acids of digestive gland tissue of L. brevis fed on fish, crustaceans or a mixed diet (Sections, F, C, Mixed). (c) and (d) Selected fatty acids of prey species used for feeding. Positive bars indicate standard deviation. The number of squid equals 14 animals per feeding group. Prey numbers analysed: Silverside=6, sheepshead minnow=45, grass shrimp=76, sailfin molly=4, striped mullet=3. Data are expressed as average normal area percentage () +standard deviation. 35 Fish prey SF Crustacean prey SC : 18:1* 18:2n-6 2:4n-6 2:5n-3 22:5n-3 22:6n-3 Fig. 2. Selected fatty acids for digestive gland of L. brevis fed on fish (SF, n=6) and after a diet swap on crustaceans (SC, n=8). Fish prey shows pooled fatty acid data of the 4 fish species used as feed. Crustacean prey shows fatty acid data of grass shrimp. Data are expressed as average normal area percentage () +standard deviation.

10 6 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) a.) 4 F 22:6n-3 Muscle DG b.) 4 C 22:6n-3 Muscle DG start day day 2 day 3 F start day day 2 day 3 C c.) F 18:2n-6 Muscle DG d.) C 18:2n-6 Muscle DG start day day 2 day 3 F start day day 2 day 3 C e.) 2 15 F 2:4n-6 Muscle DG f.) 2 15 C 2:4n-6 Muscle DG 5 5 start day day 2 day 3 F start day day 2 day 3 C Fig. 3. Changes in fatty acids 22:6 n-3 (graphs a and b), 18:2 n-6 (graphs c and d) and 2:4 n-6 (graphs e and f), over time. Data are given for muscle and digestive gland tissue (DG) in L. brevis fed on fish (F, graphs a., c., e.) and L. brevis fed on crustaceans (C, graphs b., d., f.). Data on fish prey (F) were pooled from all fish species fed in the experiment; crustacean prey (C) denotes grass shrimp. Data are expressed as average normal area percentages () from the start (F, n=2, C, n=1) and after (F, n=3, C, n=4), 2 (F and C, n=5) and 3 (F and C, n=5) days into the experiment. Positive bars indicate standard deviation. (feeding categories F and C). For example, after ten days of feeding on a crustacean diet (C), the comparatively low amounts of PUFA 22:6 n-3 in squid tissues corresponded to the low levels of this fatty acid in crustacean prey (Fig. 3a,b). From day to day 3, changes in amounts of individual fatty acids in squid tissues were less pronounced. Linoleic acid (18:2 n-6) was found in elevated levels in squid throughout the experiment, corresponding to relatively high levels in both prey types. However squid taken after 2 and 3 days showed comparatively lower levels of linoleic acid in their tissues than squid taken at day (Fig. 3c,d). On the other hand, the amount of arachidonic acid (2:4 n- 6) in squid tissues increased over the course of the experiment, with levels substantially higher than found in either prey type (Fig. 3e,f). Temporal trends in n-3/ n-6 ratios were more apparent in muscle tissue than in digestive gland. As for individual fatty acids, the highest changes in muscle occurred within the first ten days of the experiment. Ratios decreased more slowly over the next 2 days until, at the end of the experiment, the average ratio reached similar values for all sections. However, squid feeding on fish always showed slightly higher ratios than squid feeding on crustaceans (Table 2b). In digestive gland tissue, temporal trends in n-3/ n-6 ratios were found only between the start of the experiment and day, for squid fed on a crustacean diet.

11 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) a.) Carbon δ 13 C b.) Nitrogen δ 15 N days days Fig. 4. Mean (a) δ 13 C and (b) δ 15 N values for muscle and digestive gland of L. brevis fed on crustaceans (C) over time. Values for grass shrimp are included for comparison. =grass shrimp, =digestive gland tissue, =muscle tissue. Bars indicate±standard deviation Stable isotope analysis Carbon isotope ratios The presence of lipids in a tissue sample influences carbon isotope signatures (DeNiro and Epstein, 1977). Due to lipids not being removed prior to stable isotope analysis the interpretation of carbon isotope results was restricted to comparisons of squid fed on similar prey. Squid fed exclusively on crustaceans (C) showed changes in δ 13 C over the course of the experiment (Fig. 4a). Carbon isotope ratios for digestive gland tissue after three days of feeding were depleted relative to the ratios found in prey species. Muscle tissue yielded a slightly enriched ratio compare to prey species. In samples taken after days, however, δ 13 C values for both the digestive gland and muscle tissue of squid were considerably enriched compared to their prey (Tables 4 and 5). Measured against these earlier samples, squid sacrificed after 3 days showed even more enriched carbon levels in muscle tissue and no further changes in average δ 13 C in the digestive gland. No significant differences were found in δδ 13 C of muscle tissue between squid fed on crustaceans for 15 days (SC) and squid fed on crustaceans for 3 days (C). Squid fed solely on a fish diet (F) also showed more enriched levels for carbon with increasing time. Similar average carbon isotope ratios were found in digestive gland tissue of all squid that were fed exclusively on fish (SF and F). Similar ratios were also found for squid swapped from a fish to a crustacean diet (SC) and squid fed on a mixed diet. In muscle tissue, average carbon isotope ratios were closest between squid fed exclusively on crustaceans in feeding groups SC and C Nitrogen isotope ratios δ 15 N values differed significantly between wild and experimentally fed squid (ANOVA, muscle, pb.1, digestive gland, pb.1, Fig. 5a,b). δ 15 N Table 4 Isotopic composition (δ 13 C and δ 15 N) of muscle and digestive gland of L. brevis at the start, middle and end of the feeding experiment Feeding sections Sampling time Digestive gland Muscle n δ 13 C[ ] δ 15 N[ ] n δ 13 C[ ] δ 15 N[ ] Wild Day ±.67.64± ± ±.62 SF Day SC Day ± ± ± ±.52 F Day 4 and ± ± ± ±.48 F Day ± ± ± ±.3 C Day C Day ± ± ± ±1.57 C Day ± ± ±.26.98±.54 Mixed Day 3, 4 and ± ± ± ±1. Mixed Day ± ± ± ±1.26 Given are means ±standard deviation. Feeding sections: SF=switched diet/fish (day 1 15), SC=switched diet/crustacea (day 16 3), F=all fish (3 days), C = all crustacea (3 days), Mixed=fish and crustacea (3 days).

12 8 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) Table 5 Isotopic composition (δ 13 C and δ 15 N) of whole specimens of putative prey species of L. brevis Species n δ 13 C[ ] δ 15 N[ ] Silverside ± ±1.11 Sailfin molly ± ±1.4 Sheepshead minnow ± ±.36 Grass shrimp ± ±.33 Atlantic croaker ± ±2.16 Sand trout Menhaden Penaeid shrimp Given are means±standard deviation. values for muscle tissue were on average 5 higher than those for the digestive gland of the same squid. δ 15 N values were highest for the wild group and for squid dying at the start of the experiment (Table 4). In contrast to prey species taken from bay waters, fish and crustacean prey used for feeding showed similar δ 15 N values to each other (Table 5). No significant difference was found in nitrogen isotope ratios of digestive gland between squid feeding on fish (SF and F) and squid feeding on a mixed diet. Squid feeding on crustaceans (SC and C) showed the lowest δ 15 N values. δ 15 N values for muscle tissue were also higher in squid feeding on fish than in squid feeding on crustaceans. Squid feeding on a mixed diet showed intermediate δ 15 N values in their muscle tissue. Squid tissues were always enriched in δ 15 N relative to the diet. After 3 days of feeding, δ 15 N values of muscle and digestive gland were 8 higher than in grass shrimp. δ 15 N values of squid sacrificed after days were distinctly lower, and much closer to prey δ 15 N in the case of digestive gland tissue. No further significant change in nitrogen isotope ratios was seen over the next twenty days. Comparing nitrogen isotope ratios in the digestive gland of the predator (day to 3) with those in their prey, the average trophic distance from predator to prey is 1.5 for these species. The average δ 15 N level for muscle tissue was slightly different between day 3 and day, with even lower levels measured at day 3 (Table 4). However δ 15 N of squid muscle tissue remained approximately 6 higher than δ 15 N found in prey. Nitrogen isotope ratios were similar for both tissues in samples taken from group SC and samples from group C after 15 and 3 days, respectively. Squid fed solely on a fish diet (F) showed depleted levels for nitrogen with increasing time. Differences in δ 15 N values measured at the beginning and at the end of the experiment were 3 for digestive gland and 1.5 for muscle tissue. Due to the lack of 3-day data (as all specimens of group F died prior to this sampling date) for squid fed on fish, further change with time could not be investigated for this group. 4. Discussion In this study we have demonstrated that fatty acid and stable isotope signatures of squid tissues converge towards dietary signatures, with significant changes apparent days after the commencement of feeding on experimental diets. Short-term and long-term effects of the diet on squid can be detected through the analysis of metabolically a.) Digestive gland Squid feeding groups Prey b.) Muscle Squid feeding groups Prey δ 15 N (Digestive gland) δ 15 N (Muscle) Wild SF SC F C Mixed diet Estuarine fish Grass shrimp Bay fish Bay crustacean 2 Wild SF SC F C Mixed diet Estuarine Fish Grass shrimp Bay fish Bay crustacean Fig. 5. Distribution of stable nitrogen isotope ratios in (a) digestive gland and (b) muscle of L. brevis, and in prey species.

13 G. Stowasser et al. / Journal of Experimental Marine Biology and Ecology 333 (26) different tissues. The present study indicates that, by applying these methods to field data, fatty acid and stable isotope profiles could be used to reconstruct the diet composition of squid in the wild to the extent of identifying the feeding habitat and broad prey type. Bioconversion and/or selective retention of fatty acids and stable isotopes does, to some extent, occur in digestive gland and muscle tissue of squid since, after 4 weeks of feeding on a constant diet, most squid fatty acid and stable isotope signatures were still readily distinguishable from their prey, especially in muscle. These species-specific signatures suggest that fatty acid and stable isotope data could be used to indicate the presence of L. brevis in the diet of species from higher trophic levels Fatty acids Squid fed on different diets exhibited clear preyrelated fatty acid signatures in both digestive gland and muscle tissue. Prominent fatty acids typical for the fish and crustacean species given as food were shown to increase in the predators' tissues as early as ten days after commencement of experimental feeding. Navarro and Villanueva (23) also found ten days of rearing sufficient to shift fatty acids of Octopus vulgaris from a natural profile towards that of an experimental diet. Changes in relative amounts of individual fatty acids present in squid tissues were, in general, proportional to the magnitude of initial differences between prey and predator, at least for squid sacrificed during the experiment. Squid dying of natural causes showed less pronounced differences between feeding groups and less pronounced shifts in their fatty acid composition towards prey fatty acid signatures. This is likely to have been due to a combination of tissue breakdown, i.e. lipid oxidation after death (Hardy, 198), and the likelihood that these animals did not feed for some time prior to their death. Fatty acid signatures changed more rapidly in digestive gland tissue and the variation in proportions of individual fatty acids remained higher than in muscle tissue. The small range of variation between muscle samples could be because mantle tissue has a structural role, for which retention of specific fatty acids is necessary (e.g. Sargent et al., 1989). The digestive gland on the other hand is a storage organ, where fatty acids from prey accumulate mainly unchanged over a relative short period of time, thus primarily reflecting dietary intake (e.g. Phillips et al., 21). High variability in fatty acid signatures of the digestive gland found in this study thus indicates rapid turnover of the tissue and little modification of fatty acids between prey and predator. Unusual observations were noted for fatty acids 18:2 n-6 and 2:4 n-6. Both fish and crustacean prey showed high levels of 18:2 n-6 which, on the basis of changes found for other fatty acids, would be expected to cause a steady increase in proportion in squid tissues over the course of the experiment. Levels of 2:4 n-6, on the other hand, showed similar proportions in fish and crustacean prey and in squid taken from the wild. We would therefore expect changes to have been minimal or values to increase slightly if this fatty acid is accumulated in the tissue. However, after an initial increase in 18:2 n-6 in the digestive gland, levels decreased constantly over time, while proportions of 2:4 n-6 showed a constant increase over time, to levels much higher than would be expected from values found in prey species. Both these fatty acids are essential to growth, either directly or as precursors for the synthesis of higher polyunsaturated fatty acids through desaturation and chain elongation (Cowey and Sargent, 1972). The decrease of 18:2 n-6 in the digestive gland, despite the high proportions provided by the prey, and the increase of 2:4 n-6 beyond levels found in prey, suggests that squid might have the ability of bioconversion of essential fatty acids or are capable of selective uptake and retention of certain fatty acids. This is also supported by the fact that fatty acid profiles of squid, although significantly altered in the direction of prey profiles, could still be readily distinguished from their prey after four weeks of feeding, especially in the case of muscle tissue. There have been no studies to date of the ability of cephalopods to use shorter chained PUFAs and convert them into longer chained PUFAs. Abalone and fish however have been found to do so in cases of PUFA deficiencies (e.g. Kanazawa et al., 1979; Dunstan et al., 1996). For squid in which the diet was swapped from fish to crustaceans, individuals examined after the diet swap (15 days after) showed fatty acid proportions more closely resembling the new diet of crustaceans and tissues of squid fed on a mixed diet. Despite a reduction of levels in the diet, 22:5 n-3 retained its proportion in muscle tissue, again suggesting synthesis or specific retention of this fatty acid. Freshwater and marine food webs are characterised by different levels of specific fatty acids, particularly long-chain PUFAs (Hilditch and Williams, 1964). PUFA of the n-6 series are significantly higher in freshwater organisms due to their mainly land-derived diet. Therefore ratios of n-3/ n-6 are substantially lower in freshwater than in marine organisms (Sargent, 1976).