Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

Experimentally Derived δ13C and δ15N Discrimination Factors for Gray Wolves and the Impact of Prior Information in Bayesian Mixing Models

Stable isotope analysis of diet has become a common tool in conservation research. However, the multiple sources of uncertainty inherent in this analysis framework involve consequences that have not been thoroughly addressed. Uncertainty arises from the choice of trophic discrimination factors, and for Bayesian stable isotope mixing models (SIMMs), the specification of prior information; the combined effect of these aspects has not been explicitly tested. We used a captive feeding study of gray wolves (Canis lupus) to determine the first experimentally-derived trophic discrimination factors of C and N for this large carnivore of broad conservation interest. Using the estimated diet in our controlled system and data from a published study on wild wolves and their prey in Montana, USA, we then investigated the simultaneous effect of discrimination factors and prior information on diet reconstruction with Bayesian SIMMs. Discrimination factors for gray wolves and their prey were 1.97‰ for δ¹³C and 3.04‰ for δ¹⁵N. Specifying wolf discrimination factors, as opposed to the commonly used red fox (Vulpes vulpes) factors, made little practical difference to estimates of wolf diet, but prior information had a strong effect on bias, precision, and accuracy of posterior estimates. Without specifying prior information in our Bayesian SIMM, it was not possible to produce SIMM posteriors statistically similar to the estimated diet in our controlled study or the diet of wild wolves. Our study demonstrates the critical effect of prior information on estimates of animal diets using Bayesian SIMMs, and suggests species-specific trophic discrimination factors are of secondary importance. When using stable isotope analysis to inform conservation decisions researchers should understand the limits of their data. It may be difficult to obtain useful information from SIMMs if informative priors are omitted and species-specific discrimination factors are unavailable.     

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus)

The diet-tissue discrimination factor is the amount by which a consumer’s tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.     

Changes in diets of individual Baltic ringed seals (Phoca hispida botnica) during their breeding season inferred from stable isotope analysis of multiple tissues

The stable isotope ratios (δ¹³C and δ¹⁵N) of three tissues with different metabolic rates (plasma, liver, and muscle) were used to investigate temporal variation in diet among nine individual Baltic ringed seals (Phoca hispida botnica Gmelin) from the Bothnian Bay, northeast Baltic Sea. The isotope values from plasma should reflect the most recent diet, values from liver the diet of the past weeks prior to sampling, and values from muscle should integrate diet over almost the entire breeding season of the ringed seals. In general, δ¹³C values of liver were more enriched in ¹³C than were those of either muscle or plasma, suggesting that the diet of the seals may have included a higher proportion of ¹³C‐enriched benthic prey in April. Females showed more variable δ¹³C values than males, suggesting possible gender differences in diet or in foraging locations. The differences that were apparent between females possibly reflect individual variation in the onset and duration of parturition and lactation, both of which likely restrict female foraging. Previous data from parasite infections and from alimentary tract contents of the same seals were linked to the isotope data to assist in drawing inferences about changes in the diets of individual seals.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

Variation among diets in discrimination of δC and δN in the amphipod Allorchestes compressa

Discrimination of stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) was examined for the amphipod Allorchestes compressa Dana using controlled laboratory experiments. Amphipods were fed exclusively on single diets (fresh or decomposed macroalgae or seagrass) for three weeks. Macrophyte type (i.e. seagrass, brown algae or red algae) had a greater influence on the stable isotope ratios of A. compressa than the state of decomposition of the macrophyte material. The experiments revealed that δ¹³C in A. compressa stabilised at values lower than those of the diets, which contrasts to the general assumption that consumer-diet discrimination of δ¹³C ranges from 0 to + 1‰. Amphipods fed on seagrass yielded the lowest δ¹³C values, which were 9 to 10‰ lower than their diet, while amphipods fed on macroalgae had values 2 to 4‰ lower than their diet. In addition, contrary to the general assumption that consumer-diet discrimination of δ¹⁵N ranges from + 3 to + 5‰, discrimination of δ¹⁵N was as low as − 1 and + 1 when A. compressa was fed on brown and red algae, respectively, but as high as + 3‰ when fed on seagrass. The results show that discrimination of stable isotopes of carbon and nitrogen can vary considerably depending on the food source, demonstrating that validation of assumptions about discrimination are critical for interpreting stable isotope data from field studies.     

Tissue Turnover Rates and Isotopic Trophic Discrimination Factors in the Endothermic Teleost,Pacific Bluefin Tuna (Thunnus orientalis)

Stable isotope analysis (SIA) of highly migratory marine pelagic animals can improve understanding of their migratory patterns and trophic ecology. However, accurate interpretation of isotopic analyses relies on knowledge of isotope turnover rates and tissue-diet isotope discrimination factors. Laboratory-derived turnover rates and discrimination factors have been difficult to obtain due to the challenges of maintaining these species in captivity. We conducted a study to determine tissue- (white muscle and liver) and isotope- (nitrogen and carbon) specific turnover rates and trophic discrimination factors (TDFs) using archived tissues from captive Pacific bluefin tuna (PBFT), Thunnus orientalis, 1–2914 days after a diet shift in captivity. Half-life values for ¹⁵N turnover in white muscle and liver were 167 and 86 days, and for ¹³C were 255 and 162 days, respectively. TDFs for white muscle and liver were 1.9 and 1.1‰ for δ ¹⁵N and 1.8 and 1.2‰ for δ ¹³C, respectively. Our results demonstrate that turnover of ¹⁵N and ¹³C in bluefin tuna tissues is well described by a single compartment first-order kinetics model. We report variability in turnover rates between tissue types and their isotope dynamics, and hypothesize that metabolic processes play a large role in turnover of nitrogen and carbon in PBFT white muscle and liver tissues. ¹⁵N in white muscle tissue showed the most predictable change with diet over time, suggesting that white muscle δ ¹⁵N data may provide the most reliable inferences for diet and migration studies using stable isotopes in wild fish. These results allow more accurate interpretation of field data and dramatically improve our ability to use stable isotope data from wild tunas to better understand their migration patterns and trophic ecology.     

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

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 30 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 10 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.     

Comparative feeding ecology of shortfin mako, blue and thresher sharks in the California Current

This study describes the feeding ecology of three pelagic shark species in the California Current: shortfin mako (Isurus oxyrinchus); blue (Prionace glauca); and thresher (Alopias vulpinus) sharks. Stomach contents of sharks collected from 2002 to 2008 were identified to the lowest taxonomic level and analyzed using univariate and multivariate methods. Of 330 mako sharks sampled (53 to 248?cm fork length [FL]), 238 stomachs contained 42 prey taxa, with jumbo squid (Dosidicus gigas) and Pacific saury (Cololabis saira) representing the most important prey based on the geometric index of importance (GII). In addition, 158 blue sharks were sampled (76 to 248?cm FL) and 114 stomachs contained 38 prey taxa, with jumbo and Gonatus spp. squids representing the most important prey. Lastly, 225 thresher sharks were sampled (108 to 228?cm FL) and 157 stomachs contained 18 prey taxa with northern anchovy (Engraulis mordax) and Pacific sardine (Sardinops sagax) identified as the most important prey. Overall, mako sharks had the most diverse diet based upon Simpson??s diversity index (1/D) (8.43?±?1.16), feeding on many species of teleosts and cephalopods, followed by blue sharks (6.20?±?2.11) which consumed a wide range of prey (primarily cephalopods), while thresher sharks were most specialized (2.62?±?0.34), feeding primarily on coastal pelagic teleosts. Dietary overlap was lowest between blue and thresher sharks (S?rensen similarity index?=?0.321 and Simplified Morisita Horn index?=?0.006), and seasonal variability in diet was greatest for blue sharks (Simplified Morisita Horn index?=?0.260, Analysis of Similarity (ANOSIM) p?<?0.001). In addition, size class, and subregion were significant factors that affected diet of each species differently (ANOSIM p?<?0.001). Despite similarities in life history characteristics and spatial and temporal overlap in habitat, diets of these three common shark species are distinct in the California Current.     

What is the invasiveness of <Emphasis Type="Italic">Hemimysis anomala</Emphasis> (Crustacea,Mysidae) in the large deep Lake Bourget,France?

Application of stable isotope data to trophic studies requires understanding of factors influencing the isotopic discrimination factor (Δ) between consumers and their prey resources. This is missing for many omnivorous species, despite their diet and environment potentially impacting Δ. The effects of temperature, diet (including formulated feeds) and tissue type on Δ¹³C and Δ¹⁵N were thus tested experimentally. A temperature experiment exposed three species to identical diets at 18 and 23°C, whereas a diet experiment exposed one species to four diets at 18°C. At 23°C, C:N ratios, Δ¹³C and Δ¹⁵N were generally elevated versus 18°C. After lipid correction, tissue/species-specific differences at 23°C in Δ¹³C and Δ¹⁵N were up to 0.73 and 0.54‰ higher, respectively. Across the four diets, there were also significant differences in Δ¹³C and Δ¹⁵N between a natural diet and diets based on formulated feeds. Δ¹³C and Δ¹⁵N of muscle were 1.51 to 2.76‰ and 3.13 to 5.44‰, respectively. Highest Δ for both isotopes was from a formulated feed based on plant material that resulted in lower dietary protein content and quality. Thus, diet and environment fundamentally affected the isotopic discrimination factors and these factors require consideration within trophic studies based on stable isotopes.     

Trophic ecology of juvenile bull sharks (Carcharhinus leucas) in the Coyote estuary,Costa Rica

Bull shark (Carcharhinus leucas) is a near-threatened elasmobranch species capable of moving between the fresh and salty waters of tropical and subtropical coastal areas, for which we still lack important ecological information. During their first years of life, bull sharks use estuarine systems as nursery areas, making them highly susceptible to environmental and anthropogenic pressures. We studied the trophic ecology of juveniles found in the Coyote estuary, a potential nursery area in Costa Rica, to understand the potential impact of further bull shark declines and gain knowledge that could aid in their conservation. We analysed the trophic ecology of juvenile bull sharks [81–103 cm total length (TL)] in the Coyote estuary, Costa Rica, using stable isotopes of δ¹⁵N and δ¹³C. Since one problem using this technique in juveniles is the confounding effect of the maternal signature, we sampled different tissues (muscle and plasma), verified the status of the shark's umbilical scar and identified the size at which the isotope signature is a result of the animal's current diet. The isotopic values of the muscle tissue reflected the maternal isotopic signature. In contrast, plasma values reflected the diet of juvenile bull sharks >95 cm TL and with a closed umbilical scar. Juvenile bull sharks fed primarily on teleost fishes of the order Anguilliformes and Siluriformes, and have a high trophic position (≥4.0) in the Coyote estuary. Our findings suggest that this estuary is an important feeding site for juvenile bull sharks of the Pacific of Costa Rica. Thus, the protection of essential habitats such as the Coyote estuary will benefit not only bull shark conservation, but also the conservation of an array of fish species that also use this habitat as a rookery, many of which are of commercial interest.     

The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks Comment on: stable isotope dynamics in elasmobranch fishes

Stable isotopes of nitrogen and carbon (δ¹⁵N and δ¹³C) provide an important tool to examine diet, trophic position and movement/migration of both aquatic and terrestrial animals. Over the past 10 years, there have been repeated calls to tighten up basic assumptions when applying stable isotopes, one of the most important being the application of accurate, species-specific diet-tissue discrimination factors (DTDFs). Taxa- or species-specific DTDFs are required for (i) predicting dietary sources to a consumer using stable isotope mixing models and (ii) for estimating trophic position relative to primary consumers or known base species. Logan & Lutcavage (2010) recently presented data on stable isotope dynamics in elasmobranch fishes and concluded that DTDFs for teleost fish were suitable for elasmobranch fish, endorsing the generally applied value of 3.4‰. When considering (i) a recent study which found that DTDFs were lower for large sharks than teleost fish (Hussey et al., 2010) and (ii) that the Logan and Lutcavage study did not experimentally address the issue of DTDFs, we would argue that this conclusion is misleading. We demonstrate this point by estimating the proportion of prey items of a captive shark with a known diet history by modelling the δ¹⁵N values of the shark and its prey. The often repeated implication of inaccurate DTDFs is clear, with model results highly variable depending on the selected DTDF. In addition, model results for the standard teleost DTDF of 3.4‰ provided erroneous estimates of prey consumption. The suggestion that DTDFs for teleost fish are suitable for elasmobranchs may mislead investigators to choose DTDFs which are likely not applicable to their study species. Caution is therefore warranted in advocating this approach. Continued experimental work to examine stable isotopes in sharks is required and recommendations are made.     

Isotopic Incorporation Rates and Discrimination Factors in Mantis Shrimp Crustaceans

Stable isotope analysis has provided insights into the trophic ecology of a wide diversity of animals. Knowledge about isotopic incorporation rates and isotopic discrimination between the consumer and its diet for different tissue types is essential for interpreting stable isotope data, but these parameters remain understudied in many animal taxa and particularly in aquatic invertebrates. We performed a 292-day diet shift experiment on 92 individuals of the predatory mantis shrimp, Neogonodactylus bredini, to quantify carbon and nitrogen incorporation rates and isotope discrimination factors in muscle and hemolymph tissues. Average isotopic discrimination factors between mantis shrimp muscle and the new diet were 3.0 ± 0.6 ‰ and 0.9 ± 0.3 ‰ for carbon and nitrogen, respectively, which is contrary to what is seen in many other animals (e.g. C and N discrimination is generally 0–1 ‰ and 3–4 ‰, respectively). Surprisingly, the average residence time of nitrogen in hemolymph (28.9 ± 8.3 days) was over 8 times longer than that of carbon (3.4 ± 1.4 days). In muscle, the average residence times of carbon and nitrogen were of the same magnitude (89.3 ± 44.4 and 72.8 ± 18.8 days, respectively). We compared the mantis shrimps’ incorporation rates, along with rates from four other invertebrate taxa from the literature, to those predicted by an allometric equation relating carbon incorporation rate to body mass that was developed for teleost fishes and sharks. The rate of carbon incorporation into muscle was consistent with rates predicted by this equation. Our findings provide new insight into isotopic discrimination factors and incorporation rates in invertebrates with the former showing a different trend than what is commonly observed in other animals.     

Validating the Incorporation of 13C and 15N in a Shorebird That Consumes an Isotopically Distinct Chemosymbiotic Bivalve

The wealth of field studies using stable isotopes to make inferences about animal diets require controlled validation experiments to make proper interpretations. Despite several pleas in the literature for such experiments, validation studies are still lagging behind, notably in consumers dwelling in chemosynthesis-based ecosystems. In this paper we present such a validation experiment for the incorporation of ¹³C and ¹⁵N in the blood plasma of a medium-sized shorebird, the red knot (Calidris canutus canutus), consuming a chemosymbiotic lucinid bivalve (Loripes lucinalis). Because this bivalve forms a symbiosis with chemoautotrophic sulphide-oxidizing bacteria living inside its gill, the bivalve is isotopically distinct from ‘normal’ bivalves whose food has a photosynthetic basis. Here we experimentally tested the hypothesis that isotope discrimination and incorporation dynamics are different when consuming such chemosynthesis-based prey. The experiment showed that neither the isotopic discrimination factor, nor isotopic turnover time, differed between birds consuming the chemosymbiotic lucinid and a control group consuming a photosynthesis-based bivalve. This was true for ¹³C as well as for ¹⁵N. However, in both groups the ¹⁵N discrimination factor was much higher than expected, which probably had to do with the birds losing body mass over the course of the experiment.     

Diet Reconstruction and Resource Partitioning of a Caribbean Marine Mesopredator Using Stable Isotope Bayesian Modelling

The trophic ecology of epibenthic mesopredators is not well understood in terms of prey partitioning with sympatric elasmobranchs or their effects on prey communities, yet the importance of omnivores in community trophic dynamics is being increasingly realised. This study used stable isotope analysis of ¹⁵N and ¹³C to model diet composition of wild southern stingrays Dasyatis americana and compare trophic niche space to nurse sharks Ginglymostoma cirratum and Caribbean reef sharks Carcharhinus perezi on Glovers Reef Atoll, Belize. Bayesian stable isotope mixing models were used to investigate prey choice as well as viable Diet-Tissue Discrimination Factors for use with stingrays. Stingray δ¹⁵N values showed the greatest variation and a positive relationship with size, with an isotopic niche width approximately twice that of sympatric species. Shark species exhibited comparatively restricted δ¹⁵N values and greater δ¹³C variation, with very little overlap of stingray niche space. Mixing models suggest bivalves and annelids are proportionally more important prey in the stingray diet than crustaceans and teleosts at Glovers Reef, in contrast to all but one published diet study using stomach contents from other locations. Incorporating gut contents information from the literature, we suggest diet-tissue discrimination factors values of Δ¹⁵N ≊ 2.7‰ and Δ¹³C ≊ 0.9‰ for stingrays in the absence of validation experiments. The wide trophic niche and lower trophic level exhibited by stingrays compared to sympatric sharks supports their putative role as important base stabilisers in benthic systems, with the potential to absorb trophic perturbations through numerous opportunistic prey interactions.     

Terrestrial and marine trophic pathways support young-of-year growth in a nearshore Arctic fish

River discharge supplies nearshore communities with a terrestrial carbon source that is often reflected in invertebrate and fish consumers. Recent studies in the Beaufort Sea have documented widespread terrestrial carbon use among invertebrates, but only limited use among nearshore fish consumers. Here, we examine the carbon source and diet of rapidly growing young-of-year Arctic cisco (Coregonus autumnalis) using stable isotope values (δ¹³C and δ¹⁵N) from muscle and diet analysis (stomach contents) during a critical and previously unsampled life stage. Stable isotope values (δ¹⁵N and δ¹³C) may differentiate between terrestrial and marine sources and integrate over longer time frames (weeks). Diet analysis provides species-specific information, but only from recent foraging (days). Average δ¹³C for all individuals was ?25.7 ‰, with the smallest individuals possessing significantly depleted δ¹³C values indicative of a stronger reliance of terrestrial carbon sources as compared to larger individuals. Average δ¹⁵N for all individuals was 10.4 ‰, with little variation among individuals. As fish length increased, the proportion of offshore Calanus prey and neritic Mysis prey increased. Rapid young-of-year growth in Arctic cisco appears to use terrestrial carbon sources obtained by consuming a mixture of neritic and offshore zooplankton. Shifts in the magnitude or phenology of river discharge and the delivery of terrestrial carbon may alter the ecology of nearshore fish consumers.     

Plastic changes in tadpole trophic ecology revealed by stable isotope analysis

Amphibian larvae constitute a large fraction of the biomass of wetlands and play important roles in their energy flux and nutrient cycling. Interactions with predators and competitors affect their abundance but also their foraging behaviour, potentially leading to non-consumptive cascading effects on the whole trophic web. We experimentally tested for plastic changes in larval trophic ecology of two anuran species in response to competitors and the non-lethal presence of native and non-native predators, using stable isotope analysis. We hypothesized that tadpoles would alter their diet in the presence of competitors and native predators, and to a lesser extent or not at all in the presence of non-native predators. First, we conducted a controlled diet experiment to estimate tadpole turnover rates and discrimination factors using Pelobates cultripes and Bufo calamita. Turnover rates yielded a half-life of 15–20 days (attaining a quasi-isotopic equilibrium after 2 months), whereas discrimination factors for natural controlled diets resulted in different isotopic values essential for calibration. Second, we did an experiment with P. cultripes and Rana perezi (=Pelophylax perezi) where we manipulated the presence/absence of predators and heterospecific tadpoles using microcosms in the laboratory. We detected a significant shift in trophic status of both amphibian species in the presence of non-native crayfish: the δ¹⁵N values and macrophyte consumption of tadpoles increased, whereas their detritus consumption decreased. This suggests that tadpoles could have perceived crayfish as a predatory risk or that crayfish acted as competitors for algae and zooplankton. No dietary changes were observed in the presence of native dragonflies or when both tadpole species co-occurred. Stable isotopic analysis is an efficient way to assess variation in tadpoles’ tropic status and hence understand their role in freshwater ecosystems. Here we provide baseline isotopic information for future trophic studies and show evidence for plastic changes in tadpoles’ use of food resources under different ecological scenarios.     

Fishery and Ontogenetic Driven Changes in the Diet of the Spiny Dogfish, Squalus acanthias, in Patagonian Waters, Argentina

The diet of the spiny dogfish in northern and central Patagonian waters was studied from the by-catch of the bottom trawling fisheries directed to Argentine hake Merluccius hubbsi and Argentine red shrimp Pleoticus muelleri between 1996 and 1998. Food habits were analyzed in relation to sex and maturity of sharks. Also, the relationship between diet and abundance of the main prey was explored. Intraspecific diet comparisons were performed. Differences in diet were found between immature individuals, mature males, and mature females. The Argentine shortfin squid Illex argentinus, the Argentine hake and ctenophores Pleurobrachia pileus were the most important prey for the spiny dogfish. Immature and small individuals were pelagic predators. After sexual maturity, the spiny dogfish shifted its diet. Mature and large individuals tended to reduce the consumption of ctenophores, and increase the consumption of demersal and benthic species. Previous diet studies on the spiny dogfish in 1984–1985 and 1994 indicated that the Argentine hake was the most important prey. In the present study, carried out in 1998, squid was the most important prey, followed by the hake. This diet change was related to the major decrease of hake due to overfishing and the increase of squid abundance between 1994 and 1999.     

Temperature and diet affect carbon and nitrogen isotopes of fish muscle: can amino acid nitrogen isotopes explain effects?

Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) are widely used in food-web studies to determine trophic positioning and diet sources. However in order to accurately interpret stable isotope data the effects of environmental variability and dietary composition on isotopic discrimination factors and tissue turnover rates must be validated. We tested the effects of temperature and diet on tissue turnover rates and discrimination of carbon and nitrogen isotopes in an omnivorous fish, black bream (Acanthopagrus butcheri). Fish were raised at 16 °C or 23 °C and fed either a fish-meal or vegetable feed to determine turnover rates in fish muscle tissue up to 42 days after exposure to experimental treatments. Temperature and diet affected bulk tissue δ¹⁵N turnover and discrimination factors, with increased turnover and smaller discrimination factors at warmer temperatures. Fish reared on the vegetable feed showed greater bulk tissue δ¹⁵N changes and larger discrimination factors than those reared on a fish-meal feed. Temperature and diet affected bulk tissue δ¹³C values, however the direction of effects among treatments changed. Analyses of δ¹⁵N values of individual amino acids found few significant changes over time or treatment effects, as there was large variation at the individual fish level. However glutamic acid, aspartic acid and leucine changed most over the experiment and results mirrored those of treatment effects in bulk δ¹⁵N tissue values. The results demonstrate that trophic discrimination for δ¹⁵N and δ¹³C can be significantly different than those typically used in food-web analyses, and effects of diet composition and temperature can be significant. Precision of compound-specific isotope analyses (0.9‰) was larger than our effect size for bulk δ¹⁵N diet effects (0.7‰), therefore future experimental work in this area will need to establish a large effect size in order to detect significant differences. Our results also suggest that compound-specific amino acid δ¹⁵N may be useful for determining essential and non-essential amino acids for different animals.