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.     

Challenges using stable isotopes for estimating trophic levels in marine amphipods

In marine food web studies, stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) are widely used to estimate organisms’ trophic levels (TL) and carbon sources, respectively. For smaller organisms, whole specimens are commonly analyzed. However, this “bulk method” involves several pitfalls since different tissues may fractionate stable isotopes differently. We compared the δ¹⁵N and δ¹³C values of exoskeleton versus soft tissue, in relation to whole specimens, of three common Arctic amphipods in Svalbard waters: the benthic Anonyx nugax, the sympagic (ice-associated) Gammarus wilkitzkii and the pelagic Themisto libellula. The δ¹⁵N values of the exoskeletons were significantly lower than those of the soft tissues for A. nugax (10.5 ± 0.7‰ vs. 15.7 ± 0.7‰), G. wilkitzkii (3.3 ± 0.3‰ vs. 8.3 ± 0.4‰) and T. libellula (8.6 ± 0.3‰ vs.10.8 ± 0.3‰). The differences in δ¹³C values between exoskeletons and soft tissues were insignificant, except for A. nugax (−21.2 ± 0.2‰ vs. −20.3 ± 0.2‰, respectively). The δ¹⁵N values of whole organisms were between those of the exoskeletons and the soft tissues, being similarly enriched in ¹⁵N as the exoskeletons (except G. wilkitzkii) and depleted in ¹⁵N by 1.2–3.7‰ compared to the soft tissues. The δ¹⁵N-derived TLs of the soft tissues agreed best with the known feeding preferences of the three amphipods, which suggest a potential underestimation of 0.5–1.0 TL when stable isotope analyses are performed on whole crustaceans with thick exoskeletons. The insignificant or small differences in δ¹³C values among exoskeletons, soft tissues and whole specimens, however, suggest low probability for misinterpretations of crustaceans’ primary carbon source in marine ecosystems with distinctly different δ¹³C-carbon sources.     

Effects of nutrient enrichment on C and N stable isotope ratios of invertebrates,fish and their food resources in boreal streams

Carbon and nitrogen stable isotopes are frequently used to study energy sources and food web structure in ecosystems, and more recently, to study the effects of anthropogenic stress on aquatic ecosystems. We investigated the effect of nutrient enrichment on δ¹³C and δ¹⁵N in fine (FPOM), coarse (CPOM) particulate organic matter, periphyton, invertebrates and fish in nine boreal streams in south-central Sweden. In addition, we analysed the diet of benthic consumers using stable isotope data. Increases in δ¹⁵N of periphyton (R ² = 0.88), CPOM (0.78), invertebrates (0.92) and fish (0.89) were related to nutrient enrichment. In contrast, δ¹³C signatures did not change along the nutrient gradient. Our results show that δ¹⁵N has potential as a sensitive indicator of nutrient enrichment in boreal streams. Carbon and nitrogen isotopes failed to elucidate putative diets of selected aquatic consumers. Indeed, comparison of low- and high-impact sites showed that δ¹³C of many consumers were found outside the ranges of basal resource δ¹³C. Moreover, ranges of basal resource δ¹³C and δ¹⁵N overlapped at both low and high sites, making discrimination between the importance of allochthonous and autochthonous production difficult. Our findings show that a fractionation rate of 3.4‰ is not always be appropriate to assess trophic interactions, suggesting that more studies are needed on fractionation rates along gradients of impairment. Handling editor: M. Power     

Turnover rates of nitrogen stable isotopes in the salt marsh mummichog, Fundulus heteroclitus, following a laboratory diet switch

Nitrogen stable isotopes are frequently used in ecological studies to estimate trophic position and determine movement patterns. Knowledge of tissue-specific turnover and nitrogen discrimination for the study organisms is important for accurate interpretation of isotopic data. We measured δ¹⁵ N turnover in liver and muscle tissue in juvenile mummichogs, Fundulus heteroclitus, following a laboratory diet switch. Liver tissue turned over significantly faster than muscle tissue suggesting the potential for a multiple tissue stable isotope approach to study movement and trophic position over different time scales; metabolism contributed significantly to isotopic turnover for both liver and muscle. Nitrogen diet-tissue discrimination was estimated at between 0.0 and 1.2‰ for liver and –1.0 and 0.2‰ for muscle. This is the first experiment to demonstrate a significant variation in δ¹⁵ N turnover between liver and muscle tissues in a fish species.     

Using stable isotopes to study resource acquisition and allocation in procellariiform seabirds

Some procellariiform seabirds use a dual strategy for provisioning their chicks by alternating short (ST) and long (LT) foraging trips. Parent birds gain mass during LT but they lose mass while increasing the chick feeding frequency during ST. Self-feeding during LT is crucial for the success of ST because firstly most of the energy used during ST is likely to be derived from the energy stored during LT and secondly self-feeding during ST is presumed to be negligible. Self-feeding by adult procellariiforms is thus a key issue to understand allocation processes but it is still poorly known. We tested these predictions by using the stable isotope (δ¹⁵N and δ¹³C) technique on birds’ plasma and prey with the short-tailed shearwater Puffinus tenuirostris breeding at Tasmania as a model. Parent shearwaters returning to the colony after a LT have an Antarctic/subantarctic δ¹³C signature in their plasma (−23.8‰), thus indicating that they fed in cold waters, far away from their breeding colony, for their own maintenance. Parent birds returning to the colony after a ST also have a distant Antarctic/subantarctic δ¹³C signature in their plasma (−24.3‰), thus verifying that self-feeding is negligible during ST and that birds fast at that time, using energy stores built up in cold waters. Plasma δ¹⁵N values of adults (8.8‰) indicates they mainly prey upon zooplankton-eating organisms, probably mesopelagic myctophid fishes. A simple isotopic mixing model estimates that they consume by mass 87% myctophids and 13% subantarctic krill when self-feeding. Finally and as expected, the carbon isotopic signature of chick plasma (−22.2‰) was intermediate between those of high- and low-latitude marine organisms and is thus in agreement with chicks being fed with a large diversity of prey species caught by adult birds from Antarctic to Tasmanian waters. One main consequence of this system is that reproduction of a Tasmanian species is controlled by resources available at great distances from the breeding colony that drive allocation decisions of parent birds.     

Effects of nutritional restriction on nitrogen and carbon stable isotopes in growing seabirds

When using stable isotopes as dietary tracers it is essential to consider effects of nutritional state on isotopic fractionation. While starvation is known to induce enrichment of ¹⁵N in body tissues, effects of moderate food restriction on isotope signatures have rarely been tested. We conducted two experiments to investigate effects of a 50–55% reduction in food intake on δ¹⁵N and δ¹³C values in blood cells and whole blood of tufted puffin chicks, a species that exhibits a variety of adaptive responses to nutritional deficits. We found that blood from puffin chicks fed ad libitum became enriched in ¹⁵N and ¹³C compared to food-restricted chicks. Our results show that ¹⁵N enrichment is not always associated with food deprivation and argue effects of growth on diet–tissue fractionation of nitrogen stable isotopes (Δ¹⁵N) need to be considered in stable isotope studies. The decrease in δ¹³C of whole blood and blood cells in restricted birds is likely due to incorporation of carbon from ¹³C-depleted lipids into proteins. Effects of nutritional restriction on δ¹⁵N and δ¹³C values were relatively small in both experiments (δ¹⁵N: 0.77 and 0.41‰, δ¹³C: 0.20 and 0.25‰) compared to effects of ecological processes, indicating physiological effects do not preclude the use of carbon and nitrogen stable isotopes in studies of seabird ecology. Nevertheless, our results demonstrate that physiological processes affect nitrogen and carbon stable isotopes in growing birds and we caution isotope ecologists to consider these effects to avoid drawing spurious conclusions.     

Trophic ecology of reef sharks determined using stable isotopes and telemetry

Establishing the ecological role of predators within an ecosystem is central to understanding community dynamics and is useful in designing effective management and conservation strategies. We analysed differences in the trophic ecology of four species of reef sharks (Carcharhinus melanopterus, Carcharhinus amblyrhynchos, Triaenodon obesus and Negaprion acutidens) at Ningaloo Reef, Western Australia, by analysing tissue stable isotopes (δ¹⁵N and δ¹³C). We also monitored animals using acoustic telemetry to determine long-term residency patterns in a bay at the southern end of the reef, Coral Bay. Overall, mean δ¹³C was similar among species, ranging between −10.9 and −11.8‰, suggesting a food-web dependency on coastal producers. Classification and regression tree analysis identified an effect of species on δ¹⁵N that separated C. amblyrhynchos and C. melanopterus from N. acutidens and T. obesus. For C. amblyrhynchos and C. melanopterus, animals were also divided by size classes, with smaller sharks having lower average δ¹⁵N than larger animals; this suggests that δ¹⁵N increases with size for these two species. Juvenile C. melanopterus, juvenile N. acutidens and adult T. obesus had trophic levels of 3.7, for juvenile C. amblyrhynchos and adult C. melanopterus it was 4, and adult C. amblyrhynchos had a value of 4.3. Trophic-level estimates for C. melanopterus and C. amblyrhynchos corroborate previous conclusions based on diet studies. We found no evidence for a difference in isotopic composition between resident and non-resident sharks. The lack of variation in isotopic composition was consistent with high mean residency of these species recorded using acoustic telemetry, which was 79% (±0.09 SE) of days monitored for T. obesus, followed by N. acutidens (57 ± 19.55%), C. amblyrhynchos (54 ± 13%) and C. melanopterus (33 ± 8.28%). High δ¹³C composition in reef sharks and long-term residency behaviour suggest that coastal marine reserves might provide effective conservation refuges for some species.     

Sulfur,carbon, and nitrogen stable isotope ratios in soft tissues and trophic relationships of fish from the near-shore waters of the peter the great bay in the Sea of Japan

Stable isotope ratios of sulfur (³⁴S/³²S), carbon (¹³C/¹²C), and nitrogen (¹⁵N/¹⁴N) were analyzed in the soft tissues of 12 common species of fish from the near-shore waters of the Peter the Great Bay in the Sea of Japan. The average δ¹³C values of individual species varied from −20.7‰ for planktivorous fish to −16.8‰ for benthivorous fish, reflecting the growing relative contribution of benthic primary producers to fish nutrition. The majority of the various species representatives studied can be assigned to one trophic level, as indicated by their narrow range of δ¹⁵N values (9.9 to 12.6‰). Large interspecific variations were found in the sulfur stable isotope ratios of fish (the mean δ³⁴S values ranged from 11.2 to 19.5‰). This is the result of the different contributions to fish nutrition of infaunal invertebrates that are depleted in ³⁴S due to the microbial food chain of the bottom sediments.     

Preliminary assessment of Greenland halibut diet in Cumberland Sound using stable isotopes

We provide preliminary carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope assessment of the Greenland halibut (Reinhardtius hippoglossoides) diet in Cumberland Sound, with focus on two possible prey sources: pelagic represented by capelin (Mallotus villosus) and epibenthic represented by shrimp (Lebbeus polaris). The δ¹³C for the Greenland halibut stock indicated a pelagic carbon source in Cumberland Sound while stable isotope mixing models, IsoSource and MixSIR, indicated a 99% dietary composition of capelin relative to the shrimp. The δ¹⁵N did not vary across Greenland halibut size ranges and placed them at a fourth trophic position relative to a primary herbivore. This study provides the starting point for more elaborate Cumberland Sound research on the local Greenland halibut feeding ecology by confirming pelagic feeding and establishing relative trophic position as well as identifying stable isotopes as a useful tool for the study of diet in cold water fish species.     

Comparative Feeding Ecology of Bull Sharks (Carcharhinus leucas) in the Coastal Waters of the Southwest Indian Ocean Inferred from Stable Isotope Analysis

As apex predators, sharks play an important role shaping their respective marine communities through predation and associated risk effects. Understanding the predatory dynamics of sharks within communities is, therefore, necessary to establish effective ecologically based conservation strategies. We employed non-lethal sampling methods to investigate the feeding ecology of bull sharks (Carcharhinus leucas) using stable isotope analysis within a subtropical marine community in the southwest Indian Ocean. The main objectives of this study were to investigate and compare the predatory role that sub-adult and adult bull sharks play within a top predatory teleost fish community. Bull sharks had significantly broader niche widths compared to top predatory teleost assemblages with a wide and relatively enriched range of δ¹³C values relative to the local marine community. This suggests that bull sharks forage from a more diverse range of δ¹³C sources over a wider geographical range than the predatory teleost community. Adult bull sharks appeared to exhibit a shift towards consistently higher trophic level prey from an expanded foraging range compared to sub-adults, possibly due to increased mobility linked with size. Although predatory teleost fish are also capable of substantial migrations, bull sharks may have the ability to exploit a more diverse range of habitats and appeared to prey on a wider diversity of larger prey. This suggests that bull sharks play an important predatory role within their respective marine communities and adult sharks in particular may shape and link ecological processes of a variety of marine communities over a broad range.     

Stable isotope profiles in whale shark (<Emphasis Type="Italic">Rhincodon typus</Emphasis>) suggest segregation and dissimilarities in the diet depending on sex and size

We investigated the sex- and size-related differences in the diet of whale sharks from the Arabian Sea (north-western Indian Ocean) using carbon and nitrogen stable-isotope analyses in white muscle. The samples were collected during the commercial fishing season between April and May of 2001 in Veraval (Gujarat, India). The overall isotope signature was similar to that of the pelagic-neritic zooplanktivore Ilisha melastoma, which suggests that both species are feeding on similar prey. In whale sharks, a positive relationship was found between δ¹⁵N and δ¹³C. This, together with a significant enrichment of both heavy stable isotopes with total length indicates that the contribution to the diet of small fish and/or larger zooplankton of higher trophic level increases with the movement from offshore areas to coastal areas as they grow. Gender differences in the isotopic ratios were not statistically significant, but small sample size cannot rule out completely the existence of some degree of spatial or dietary segregation between sexes.     

Contributions of stable-isotope data to elucidating food webs of Mediterranean rocky littoral fishes

The food webs of rocky infra-littoral ecosystems in the Mediterranean have been little studied. In this investigation stable isotopes and dietary data were compared in an attempt to describe features of the food webs concerned. δ¹³C and δ¹⁵N were determined for plants, invertebrates and fishes from the Bay of Calvi, Corsica. Dietary data were derived from the literature. δ¹³C of plants ranged from –8.59‰ to –33.74‰, of benthic invertebrates from –17.0‰ to –20.52‰, of planktonic invertebrates from –20.08‰ to –22.34‰ and of fishes from –16.27‰ to –19.59‰. δ¹⁵N was generally greater at higher trophic levels. δ¹⁵N of plants was 0.95–2.92‰, of benthic invertebrates 1.69–6.54‰, of planktonic invertebrates 3.51–6.82‰ and of fishes 4.63–9.77‰. ¹³C enrichment tended to be associated with benthic food chains and ¹³C depletion with planktonic chains. Stable-isotope data suggested more varied diets for many species than implied by gut-contents data. Omnivory and trophic plasticity were widespread, and many consumers fed lower down the food chain than previous studies had suggested. Both stable-isotope and gut-contents analysis resolved differences between fishes feeding on planktonic and benthic prey and indicated that the herbivorous fish Sarpa salpa fed on a diet substantially different from that of other fishes. Zooplankton were important in the diets of several consumers (both primary and secondary), as was plankton derived detritus. One species of fish previously identified as planktivorous was shown to feed largely on benthic organisms, whilst several species of benthic invertebrates may feed on plankton-derived detritus. Although herbivores seemed to obtain most of their C from macroalgae, δ¹⁵N data suggested that many of these animals supplemented their intake of N, although gut-contents analysis did not provide evidence for such uptake. The isotopic data have elucidated several features of the food web which we would not otherwise have detected. Received: 26 April 1999 / Accepted: 24 September 1999     

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.     

Feeding ecology of phocid seals and some walrus in the Alaskan and Canadian Arctic as determined by stomach contents and stable isotope analysis

Feeding habits of ringed (Phoca hispida), bearded (Erignathus barbatus), spotted (Phoca largha) and ribbon (Phoca fasciata) seals and walrus (Odobenus rosmarus) were studied using stomach contents and stable carbon and nitrogen isotopes. Bearded seals fed benthically, primarily crustaceans and mollusks. Both zooplankton and fish were significant prey for ringed seals, while fish was principal spotted seal prey. Few gastric contents were available from ribbon seals. δ¹⁵N was positively correlated with age in ribbon seals and δ¹³C was positively correlated with age in ringed and ribbon seals. δ¹⁵N was highest in spotted seals, in agreement with their fish-dominated diet. δ¹⁵N was not different between Alaskan-harvested ringed and bearded seals, while δ¹⁵N was lowest in ribbon seals and walrus. Carbon-13 was most enriched in bearded seals and walrus reflecting benthic ecosystem use. Canadian ringed seals were depleted in ¹³C compared to Alaskan pinnipeds, likely because of Beaufort Sea versus Chukchi and Bering seas influence.     

Freezing and chemical preservatives alter the stable isotope values of carbon and nitrogen of the Asiatic clam (Corbicula fluminea)

We tested the impacts of most common sample preservation methods used for aquatic sample materials on the stable isotope ratios of carbon and nitrogen in clams, a typical baseline indicator organism for many aquatic food web studies utilising stable isotope analysis (SIA). In addition to common chemical preservatives ethanol and formalin, we also assessed the potential impacts of freezing on δ¹³C and δ¹⁵N values and compared the preserved samples against freshly dried and analysed samples. All preservation methods, including freezing, had significant impacts on δ¹³C and δ¹⁵N values and the effects in general were greater on the carbon isotope values (1.3–2.2‰ difference) than on the nitrogen isotope values (0.9–1.0‰ difference). However, the impacts produced by the preservation were rather consistent within each method during the whole 1 year experiment allowing these to be accounted for, if clams are intended for use in retrospective stable isotope studies.     

Benthic-pelagic trophic interactions within the fish assemblage in the western Bering Sea shelf area according to stomach content analysis and ratios of C and N stable isotopes

The composition, abundance, diet and trophic status of zooplankton, bottom invertebrates, fish and nekton were analyzed based on the data collected by the staff of the TINRO-Center during complex bottom trawl catches on the Bering Sea shelf in the fall of 2004. The stomach contents of mass fish species were analyzed and the nitrogen and carbon isotopic composition of 36 mass species of plankton, benthos, nekton and nektobenthos, which together make up the basis of pelagic and bottom communities, was determined. It was found that zooplankton noticeably differ from benthic invertebrates in carbon isotopic composition: δ¹³C values in zooplankton varied from −20.3‰ to −17.9‰; in benthos—from −17.5‰to −13.0‰; and in fish—from −19.2‰ (juvenile walleye pollock) to −15.3‰ (saffron cod). The levels of ¹³C isotope in the tissues of fish depended mostly on the share of pelagic or benthic animals in their diet. δ¹⁵N values in the studied species ranged from 8.6‰ (in sea urchins) to 17.2‰ (in large Pacific cods), which corresponds to a trophic level of 2.8. Obviously the δ¹⁵N values reflect the degree of predation and generally show the ratio of primary, secondary and tertiary consumers in a fish’s diet. Trophic interactions manifest a high degree of interdependence between benthic and pelagic communities (even without taking into account such lower components of the food web as phytoplankton, bacteria, and protozoa) occurring in most nektonic species that depend on both bottom and pelagic food.     

Sediment organic matter in mountain lakes of north-western Slovenia and its stable isotopic signatures: records of natural and anthropogenic impacts

Sediment organic matter (OM) and its stable carbon and nitrogen isotopes were studied in 12 Slovenian mountain lakes in the Julian Alps. The lakes have different catchment areas and display a range of trophic states. Surface sediment atomic C/N ratios ranged from 8.4 to 13.2. Based on these C/N ratios, we concluded that autochthonous OM dominates in these lakes and constitutes approximately 65–92% of the total OM. Higher contributions of autochthonous OM sources were observed in lakes above the tree line. Relatively constant C/N ratios in the deeper sediments suggest that degradation processes are most intense in the upper few centimetres of the sediments and/or that remaining OM is relatively resistant to further degradation. Surface sediment δ¹³C and δ¹⁵N values ranged from −36.1 to −14.1‰ and from −5.2 to +1.1‰, respectively. In sediment cores from seven lakes, higher δ¹³C and lower δ¹⁵N values characterize oligotrophic lakes situated above the tree line, whereas the reverse is true for eutrophic lakes below the tree line that are also exposed to more anthropogenic impact. Carbon and nitrogen biogeochemical cycling differs considerably among the lakes. Stratigraphic shifts in carbon, total nitrogen, C/N ratios and stable C and N isotopes in cores record changes in inputs, and hence water column processes, as well as alterations in loading to the lakes. The stratigraphic variations are also the result of post-depositional diagenetic changes in the upper few centimetres of sediment. All the lakes show impacts from recent increases in atmospheric deposition of dissolved inorganic nitrogen. Application of sediment OM analysis thus proved to be useful to reconstruct paleoecological changes in sensitive mountain lake ecosystems that are either natural and/or anthropogenically derived.     

Influence of substratum on the variability of benthic biofilm stable isotope signatures: implications for energy flow to a primary consumer

Benthic biofilms have been identified using stable isotope analysis (SIA) as an important resource supporting many freshwater food webs. However, biofilm δ¹³C signatures are highly variable in freshwaters, which may hamper our understanding of energy flow through food webs in these systems. There has been little consideration of the influence that substratum may have on biofilm δ¹³C signature variability and energy flows to primary consumers. We investigated the effect of organic and inorganic substrata on biofilm dynamics by examining: (1) temporal variability of biofilm stable isotope (δ¹³C, δ¹⁵N) signatures on allochthonous leaf-litter (Eucalyptus camaldulensis) and cobble substrata over 12 months in a lowland river in south-eastern Australia; and (2) the effect of substrata on biofilm energy flows to a grazer snail, Physa acuta (Gastropoda: Physidae), using SIA and ecological stoichiometry in a laboratory experiment. The temporal study indicated that cobble biofilm varied significantly in δ¹³C signature during the 12 months (up to 11‰), whereas the δ¹³C signature of leaf biofilm was less variable (less than 2‰). In contrast, biofilm δ¹⁵N signatures varied temporally on both cobble (2.6‰) and leaf (1‰) substrata. This suggests that leaf biofilm was more reliant on leaf tissue for carbon and therefore less limited by carbon supply than cobble biofilm whereas for nitrogen biofilm on both substrata was reliant on external sources. In the laboratory experiment, snails fed leaf biofilm reflected more of an allochthonous δ¹³C signature than cobble biofilm fed snails, suggesting assimilation of leaf carbon via the heterotrophic microbial community within the biofilm. Snails grew largest on cobble biofilm, which had lower C:N ratios than leaf biofilm. Our results demonstrate that the type of substratum can influence the temporal variability of biofilm δ¹³C signatures and energy flow to primary consumers.     

Establishing pathways of energy flow for insect predators using stable isotope ratios: field and laboratory evidence

 Quantifying pathways of energy transfer between plants, pests, and beneficial insects is a necessary step toward maintaining pest stable agroecosystems in the absence of chemical subsidies. A diet switching experiment utilizing a predatory ladybird beetle, Hippodamia variegata (Goeze), evaluated the use of naturally occurring stable C and N isotopes as an economically feasible and safe method for quantifying pathways of energy flow within agroecosystems. Stable isotope values of the ladybird beetle Coleomegilla maculata lengi (Timberlake) collected from an agroecosystem were used to estimate the relative amount of C and N derived from agricultural plants and incorporated by ladybird beetles based on mass balance equations. At the beginning of the diet-switching experiment δ¹³C and δ¹⁵N values of H. variegata (–12.0‰ and 6.3‰, respectively) differed by –0.2‰ and 2.9‰ from the aphids that were provided exclusively as their diet. These data are consistent with previous estimates of trophic level isotope effects. After switching the diet of H. variegata to an alternative food, isotope values of H. variegata gradually shifted toward expected values for individuals fed this diet (–22.9‰ and 8.8‰ for δ¹³C and δ¹⁵N values, respectively). Isotope values of another ladybird beetle, C. maculata, collected from the field indicated that in May, alfalfa and maize (pollen) obtained in the previous year contributed 32% and 68% of the C or N to the diets of these individuals and in August, 52%, 6%, and 42% of the C or N assimilated by these insects was derived from alfalfa, wheat, and maize, respectively. These data are consistent with expectations based on the relative abundance of C. maculata in various crops during the season. The field and laboratory data are a clear indication that isotope values are sensitive to dietary changes on a relatively short time scale (days) and provide a strong basis for the use stable C and N isotope to trace energy flow patterns of these beneficial organisms within agroecosystems. Received: 17 November 1995 / Accepted: 20 June 1996     

A free-air system for long-term stable carbon isotope labeling of adult forest trees

Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in the canopy of 25 m tall forest trees. Labeling of canopy air was achieved by continuous release of CO₂ with a δ¹³C of −46.9‰. To this end, micro-porous tubes were suspended at c. 1 m distance vertically through the canopy, minimizing CO₂ gradients from the exterior to the interior and allowing for C labeling exposure during periods of low wind speed. Target for CO₂ concentration ([CO₂]) increase was ambient +100 μmol mol⁻¹. Canopy [CO₂] stayed within 10% of the target during more than 57% of the time and resulted in a drop of δ¹³C in canopy air by 7.8‰. After 19 labeling days about 50% of C in phloem sugars and stem CO₂ efflux were turned over and 20–30% in coarse root CO₂ efflux and soil CO₂. The isoFACE system successfully altered δ¹³C of canopy air for studying turn-over of C pools in forest trees and soils, highlighting their slow turn-over rates.