Comparing pellet and stable isotope analyses of nestling Bonelli's Eagle Aquila fasciata diet

Diet analyses are central to the study of avian trophic ecology, and stable isotope analyses have made an increasing contribution in the last two decades. Few isotopic studies have assessed the diet of raptor species, which are more frequently analysed by conventional diet methods such as pellet analysis. In this study, we compare prey consumption estimates of nestling Bonelli's Eagles Aquila fasciata from conventional pellet analysis (in terms of items and biomass) and stable isotopic mixing models (SIAR) using δ¹³C, δ¹⁵N and δ³⁴S of feathers. The pellet analysis showed that European Rabbits Oryctolagus cuniculus, pigeons (mainly Common Wood Pigeons Columba palumbus and Domestic Pigeons Columba livia dom.), Red‐legged Partridges Alectoris rufa, passerines, Yellow‐legged Gulls Larus michahellis and Eurasian Red Squirrels Sciurus vulgaris were the main prey, so they were selected for diet reconstructions in SIAR. At the population level, mean prey consumption estimates were similar for pellets (both items and biomass) and SIAR. At the territory level, the weighted kappa statistic showed good ordinal scale agreement in main prey consumption between items or biomass and SIAR. Although the intraclass correlation coefficient showed poor method agreement when considering all prey in the same analysis, the intraclass correlation coefficients for each prey category showed significant agreement between pellets and SIAR when estimating the consumption of Rabbits, pigeons and Gulls, with lower agreement for passerines and Squirrels. Lastly, there was poor method agreement for estimates of Partridges. Our results suggest an overall agreement between the pellet analysis and SIAR when estimating nestling Bonelli's Eagle diet at both the population and, to a lesser extent, the territory level, supporting the usefulness of isotopic mixing models when identifying the terrestrial and marine components of raptor diets.     

Immune function and survival of great tit nestlings in relation to growth conditions

Life history theory predicts a trade-off between number and quality of offspring. Reduced quality with increasing brood size may arise from a decrease in body condition or in immunocompetence that would be important in fighting off virulent parasites by immunologically naive offspring. We tested the effect of rearing conditions on immune function of nestling great tits (Parus major) by reducing or increasing broods by two hatchlings. In the middle of the nestling period (on day 8), nestlings from enlarged broods developed lower T cell responses [as measured from the cutaneous swelling reaction to injection with phytohaemagglutinin (PHA)] and tended to have lower total leukocyte and lymphocyte concentrations in their peripheral blood than nestlings from reduced broods. Brood size manipulation affected the PHA response of nestlings most strongly in small clutches, suggesting that nestling immune function was dependent on their parents’ condition, as estimated by original clutch size. Intra-brood differences in nestling mortality were unrelated to immune parameters, but nestlings in broods without mortality had a stronger PHA response, higher concentration of lymphocytes and higher body mass on day 15 than nestlings in broods with mortality. These results support the prediction that the immune function of altricial birds is affected by rearing conditions, and that growth and immune parameters are related to inter-brood differences in nestling survival. Received: 1 February 1999 / Accepted: 19. July 1999     

Selection for fast and slow exploration affects baseline and stress-induced corticosterone excretion in Great tit nestlings, Parus major

In nestlings, glucocorticoid (GC) secretion has short-term and long-term f itness consequences. For example, short-time elevations trigger begging activity, whereas chronically elevated GC levels impair body condition, growth and cognitive abilities. Despite a growing body of literature on personality traits, the effects of selection for fast and slow exploration on GC secretion have received little attention. We compared baseline and stress-induced hypothalamic–pituitary–adrenal (HPA) axis activity of hand-reared great tit nestlings of lines selected for fast and slow exploration. Nestling droppings were collected under three conditions: control, test (following handling stress, day 14 after hatching) and the following day. The concentrations of excreted immunoreactive corticosterone metabolites (CM) were determined via an enzyme immunoassay. We also observed nestlings' begging behaviour. CM differed significantly between the lines. Nestlings of the fast line excreted lower CM than slow-line birds. In response to handling stress, nestlings excreted significantly higher concentrations of CM than during the control and on the day after handling. Sex and begging activity were not related to CM levels. Under the control condition, but not after handling, males begged significantly more often than females. In both lines, adults excreted significantly less CM compared to nestlings. Both nestlings and adults of the slow line produced higher baseline CM values than fast-line birds. Fast-line nestlings excreted lower baseline CM than nestlings of a wild population not selected for fast or slow exploration. Slow-line nestlings did not. Our results show that selection on the basis of exploratory behaviour affected HPA axis reactivity.     

Evaluating the utility of stable isotope analyses of archived freshwater sample materials

We evaluated the potential utility of stable isotope analysis of tissues commonly archived by aquatic biologists. Previous studies with chemically preserved samples have shown contradictory results, which present an obstacle for the use of archived sample materials. We tested the effects of ethanol and formalin preservation on zooplankton and of ethanol on benthic macroinvertebrate δ¹³C and δ¹⁵N values. We found that neither formalin nor ethanol had a significant effect on δ¹³C and δ¹⁵N values of preserved zooplankton. Nor did ethanol significantly affect δ¹³C or δ¹⁵N values of macroinvertebrates. However, ethanol preservation slightly, but significantly decreased C:N ratios of both zooplankton and macroinvertebrates, probably reflecting some extraction of lipids. Overall, the effects of preservatives on δ¹³C and δ¹⁵N values that we observed were minor. We also compared δ¹³C and δ¹⁵N values analysed from roach scales and perch operculum bones with those analysed from muscle tissue. Decalcification of scales and operculum bones only slightly improved our comparison to muscle tissue δ¹³C and δ¹⁵N values. Decalcified scales had slightly higher δ¹³C and lower δ¹⁵N values. Similarly, decalcified operculum bones showed slightly increased δ¹³C and decreased δ¹⁵N values to those for fish muscle. Our results confirm that scales and operculum bones can provide a suitable proxy for fish muscle in isotope studies with minor correction. We conclude that various archived sample materials can indeed be used with confidence for historical reconstructions of freshwater food webs by stable isotope analysis. Handling editor: K. Martens     

Combining sources in stable isotope mixing models: alternative methods

Stable isotope mixing models are often used to quantify source contributions to a mixture. Examples include pollution source identification; trophic web studies; analysis of water sources for soils, plants; or water bodies, and many others. A common problem is having too many sources to allow a unique solution. We discuss two alternative procedures for addressing this problem. One option is a priori to combine sources with similar signatures so the number of sources is small enough to provide a unique solution. Aggregation should be considered only when isotopic signatures of clustered sources are not significantly different, and sources are related so the combined source group has some functional significance. For example, in a food web analysis, lumping several species within a trophic guild allows more interpretable results than lumping disparate food sources, even if they have similar isotopic signatures. One result of combining mixing model sources is increased uncertainty of the combined end-member isotopic signatures and consequently the source contribution estimates; this effect can be quantified using the IsoError model (). As an alternative to lumping sources before a mixing analysis, the IsoSource mixing model () can be used to find all feasible solutions of source contributions consistent with isotopic mass balance. While ranges of feasible contributions for each individual source can often be quite broad, contributions from functionally related groups of sources can be summed a posteriori, producing a range of solutions for the aggregate source that may be considerably narrower. A paleohuman dietary analysis example illustrates this method, which involves a terrestrial meat food source, a combination of three terrestrial plant foods, and a combination of three marine foods. In this case, a posteriori aggregation of sources allowed strong conclusions about temporal shifts in marine versus terrestrial diets that would not have otherwise been discerned.     

An assessment of sample processing methods for stable isotope analyses of marine food webs

Carbon and nitrogen stable isotope ratios are commonly used in the study of marine food webs. However, different sample processing methods can influence the measurement of these stable isotope ratios. The purpose of this study is to define an adequate methodology to be used in the construction of whole food webs. It is demonstrated that acidification of the samples results in a decrease in carbon stable isotope values for sedimentary organic matter, suspended particulate organic matter, plankton and invertebrates with carbonated structures. The response was variable for nitrogen isotope ratios. Based on our results we recommend sample acidification for carbon analysis in these compartments where effects of this treatment were observed. We observed a decrease in δ¹³C values after washing with distilled water, so we do not recommend washing with water after acidification. For nitrogen analysis, acidification should be avoided. The various dehydration treatments studied caused significant differences only in nitrogen isotope ratios.     

Caterpillar abundance in the territory affects the breeding performance of great tit Parus major minor

The effects of caterpillar food supply on the breeding performance of a population of the Japanese great tit Parus major minor were investigated. Since more than 90% of the food items in our study site were caterpillars living on trees, we estimated the food availability using 20 frass traps per hectare. The sampling error of this method was about 10% on average, which was accurate enough to detect differences between territories. Food abundance at laying in each territory affected the timing of egg laying. However, food amount after hatching was correlated with clutch size. No relationship was found between fledgling quality and food availability, probably because the effects of local variation in food abundance could be canceled out by parental effort such as extending the foraging area. There was a significant negative correlation between the length of the nestling period and food availability. We suggest that parent tits decide the timing of fledging at the point where two factors, predation risk before fledging and additional improvement of nestling quality, are balanced. Food availability just after fledging affected the length of post-fledging parental care; it seems that fledglings in “poor” territories would have had difficulty in finding food and hence needed to depend on their parents longer than those in “rich” territories. Received: 10 June 1997 / Accepted: 29 December 1997     

The extended Moran effect and large-scale synchronous fluctuations in the size of great tit and blue tit populations

1. Synchronous fluctuations of geographically separated populations are in general explained by the Moran effect, i.e. a common influence on the local population dynamics of environmental variables that are correlated in space. Empirical support for such a Moran effect has been difficult to provide, mainly due to problems separating out effects of local population dynamics, demographic stochasticity and dispersal that also influence the spatial scaling of population processes. Here we generalize the Moran effect by decomposing the spatial autocorrelation function for fluctuations in the size of great tit Parus major and blue tit Cyanistes caeruleus populations into components due to spatial correlations in the environmental noise, local differences in the strength of density regulation and the effects of demographic stochasticity. 2. Differences between localities in the strength of density dependence and nonlinearity in the density regulation had a small effect on population synchrony, whereas demographic stochasticity reduced the effects of the spatial correlation in environmental noise on the spatial correlations in population size by 21.7% and 23.3% in the great tit and blue tit, respectively. 3. Different environmental variables, such as beech mast and climate, induce a common environmental forcing on the dynamics of central European great and blue tit populations. This generates synchronous fluctuations in the size of populations located several hundred kilometres apart. 4. Although these environmental variables were autocorrelated over large areas, their contribution to the spatial synchrony in the population fluctuations differed, dependent on the spatial scaling of their effects on the local population dynamics. We also demonstrate that this effect can lead to the paradoxical result that a common environmental variable can induce spatial desynchronization of the population fluctuations. 5. This demonstrates that a proper understanding of the ecological consequences of environmental changes, especially those that occur simultaneously over large areas, will require information about the spatial scaling of their effects on local population dynamics.     

Habitat structure and prey composition generate contrasting effects on carotenoid‐based coloration of great tit Parus major nestlings

Carotenoid‐based coloration of nestling plumage is generally considered a reliable signal of quality and has consistently been related to habitat structure. The main hypothesis proposed to explain this correlation is that high quality habitats contain high quality food, which in return affects the expression of carotenoid‐based plumage. It therefore assumes that, at the population level, the link between habitat structure and food composition is consistent and more relevant than inter‐individual differences in foraging ability or parental investment. In addition, it is assumed by default that food and habitat produce concordant effects on nestling coloration. In this work we evaluated habitat structure and prey composition in addition to several measures of parental investment. We investigated their relative effect on carotenoid‐based plumage coloration (lightness, chroma and hue) of great tit Parus major nestlings. We found a low correlation between carotenoid‐based coloration of nestlings and that of their parents. Nestling coloration, especially lightness and chroma, increased with the intake of more spiders. The time of breeding was positively correlated with lightness and chroma and negatively correlated with hue. Finally, the maturity of oak trees surrounding nest‐boxes correlated negatively with lightness, and the size of all tree species surrounding nest‐boxes correlated positively with hue of chick plumage. Our findings support the view that habitat structure and prey composition may produce divergent effects on feather pigmentation, and that prey proportions and variables related to parental investment should be assessed when considering carotenoid‐based coloration of chicks. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 547–555.     

The diet of ruffe Gymnocephalus cernuus (L.) in northern lakes: new insights from stable isotope analyses

The diet of ruffe Gymnocephalus cernuus was studied in two native populations in lakes of different productivity in south-west Finland using both traditional stomach content analyses and stable isotopes. According to stomach content analyses, chironomids were the most important prey type in both lakes and the diversity of the other prey reflected the zoobenthos community in the lake. Stable isotope analyses of carbon and nitrogen showed distinctive lake-specific and total length ( L _T)-related patterns in both δ¹³C and δ¹⁵N values, which could not be explained solely with diet changes. In the large mesotrophic Pyhäjärvi, both ¹³C and ¹⁵N isotopes became slightly enriched with increasing ruffe L _T, although stomach contents analyses did not suggest any diet change in larger ruffe. In the hypereutrophic Köyliönjärvi, the carbon isotope signatures of ruffe especially showed wide variation (−33·5 to −24·1‰), which was probably due to variable consumption of prey items with highly negative carbon isotope signatures. Overall, this study emphasizes that the interpretation of stable isotope results requires extensive background data of the system and that even then the diet composition of a consumer may be very difficult to define due to large variation in the signatures.     

The effect of bacterivorous nematodes on detritus incorporation by macrofaunal detritivores: A study using stable isotope and fatty acid analyses

Several studies have investigated the effect of nematodes on microbial degradation of macrophyte detritus, but little is known about the potential effect of bacterivorous nematodes on productivity of macrofaunal detritivores. We investigated the influence of the bacterivorous nematode Rhabditis (Pellioditis) mediterranea on the incorporation rate of isotopically-labelled macroalgal detritus by the amphipod Paramoera chevreuxi in a laboratory microcosm. The fatty acid composition of amphipods, nematodes, and macroalgal detritus was characterized to help determine the pathway of detritus incorporation by amphipods. The potential importance of R. mediterranea as a source of highly unsaturated fatty acids (HUFAs) to higher trophic levels was also investigated.We found no clear evidence for an effect of nematodes on the incorporation rate of fresh macroalgal detritus by amphipods, although there was some indication that the type of detritus (i.e. the green Chaetomorpha sp. vs the red Polysiphonia sp.) is important in determining the nature and extent of the relationship between nematodes and macrofaunal detritivores. Fatty acid data indicated that nematodes did not contribute significantly to the diet of amphipods when detritus was present, and there was no evidence that nematodes affected the pathway of detritus incorporation by amphipods. Amphipods incorporated Chaetomorpha sp. detritus about 10 times faster than Polysiphonia sp. detritus despite the higher C/N ratio and lower HUFA content of the former. This suggests that other factors, such as the presence of grazer deterrent compounds, are important in determining the palatability of macroalgal detritus. Amphipods fed exclusively on nematodes retained high HUFA levels but suffered high mortality. The burrowing behaviour of nematodes is suggested as the most likely factor limiting their availability to predators.Results suggest a limited interaction between amphipods and bacterivorous nematodes in detrital food webs. Further experiments are needed to test the wider applicability of these findings to different nematode and macrofaunal taxa, and for different types of detritus.     

Introduced mammals coexist with seabirds at New Island,Falkland Islands: abundance,habitat preferences,and stable isotope analysis of diet

The largest known colony of Thin-billed prions Pachyptila belcheri has been coexisting with introduced mammals for more than 100 years. Three of the introduced mammals are potential predators of adults, eggs and chicks, namely ship rats Rattus rattus, house mice Mus musculus and feral cats Felis catus. We here determine habitat preferences over three seasons and dietary patterns of the unique set of introduced predators at New Island, Falkland Islands, with emphasis on the ship rats. Our study highlights spatial and temporal differences in the levels of interaction between predators and native seabirds. Rats and mice had a preference for areas providing cover in the form of the native tussac grass Parodiochloa flabellata or introduced gorse Ulex europaeus. Their diet differed markedly between areas, over the season and between age groups in rats. During the incubation period of the prions in November–December, ship rats had mixed diets, composed mainly of plants and mammals, while only 3% of rats had ingested birds. The proportion of ingested birds, including scavenged, increased in the prion chick-rearing period, when 60% of the rats consumed prions. We used δ¹³C and δ¹⁵N to compare the importance of marine-derived food between mammal species and individuals, and found that rats in all but one area took diet of partly marine origin, prions being the most frequently encountered marine food. Most house mice at New Island mainly had terrestrial diet. The stable isotope analysis of tissues with different turnover times indicated that individual rats and mice were consistent in their diet over weeks, but opportunistic in the short term. Some individuals (12% of rats and 7% of mice) were highly specialized in marine-derived food. According to the isotope ratios in a small sample of cat faeces, rodents and rabbits were the chief prey of cats at New Island. Although some individuals of all three predators supplement their terrestrial diet with marine-derived food, the current impact of predation by mammals on the large population of Thin-billed prions at New Island appears small due to a number of factors, including the small size of rodent populations and restriction mainly to small areas providing cover. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ingrid Schenk: deceased     

Assessment of anadromous salmon resources in the diet of the Alexander Archipelago wolf using stable isotope analysis

The Alexander Archipelago wolf (Canis lupus ligoni) is unique to southeast Alaska, occurring on islands south of Frederick Sound and along the mainland between Dixon Entrance and Yakutat Bay. Sitka black-tailed deer (Odocoileus hemionus sitkensis) are an important prey species for wolves across the southern part of the region. Spawning salmon (Onchorynchus sp.) are seasonally available but their presence in wolf diets has not previously been quantified. We examined the range of bone collagen δ¹³C and δ¹⁵N values for wolves throughout southeast (n = 163) and interior (n = 50) Alaska and used a dual-isotope mixing model to determine the relative contribution of salmon-derived marine protein in the diet. Southeast Alaska wolves consumed significantly more salmon (mean ± SE: 18.3 ± 1.2%) than did wolves from interior Alaska (9.1 ± 0.6%, P<0.001). Wolves on the southeast Alaska mainland appeared to have higher marine isotopic signatures than island wolves, although this difference was not significant. Variation among individual wolf diets was higher for southeast than for interior Alaska wolves, and variation was highest in coastal mainland wolf diets (P<0.001). Marine resources may augment the diet of southeast Alaska wolves during seasonal or annual fluctuations in the availability of deer, particularly in those areas on the mainland where densities of terrestrial ungulates are relatively low. Received: 1 July 1998 / Accepted: 23 February 1999     

Using Bayesian stable isotope mixing models to estimate wolf diet in a multi-prey ecosystem

Stable isotope analysis (SIA) of wolf (Canis lupus) tissues can be used to estimate diet and intra-population diet variability when potential prey have distinct δ¹³C and δ¹⁵N values. We tested this technique using guard hairs collected from 44 wolves in 12 northwestern Montana packs, summer 2009. We used hierarchical Bayesian stable isotope mixing models to determine diet and scales of diet variation from δ¹³C and δ¹⁵N of wolves and potential prey, white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus), elk (Cervus canadensis), moose (Alces alces), snowshoe hare (Lepus americanus), and other prey. As a check on SIA results, we conducted a separate diet analysis with temporally matched scats (i.e., collected in summer 2008) from 4 of the same packs. Wolves were centered on the ungulate prey in the isotope mixing space. Both methods revealed differences among pack diets and that wolves may consume moose in greater proportions than predicted by available biomass. Stable isotope analysis, and scat results were not entirely concordant; assumptions related to tissues of use in SIA, hair growth period in wolves, and scat sampling may have contributed to a mismatch between methods. Incorrect fractionation values, insufficient separation of prey in the isotope mixing space, choice of prior information in the Bayesian mixing models, and unexplained factors may have distorted diet estimates. However, the consistently high proportion of moose in pack diets suggests that increased population monitoring would benefit management of moose and wolves. Our results also support suggestions of other researchers that species-specific fractionation values should be used whenever possible, and that SIA may sometimes only provide indices of use for general groups of prey (e.g., large ungulates). © 2012 The Wildlife Society.     

The effect of dietary protein quality on nitrogen isotope discrimination in mammals and birds

We tested the competing hypotheses that (1) nitrogen discrimination in mammals and birds increases with dietary nitrogen concentration or decreasing C:N ratios and, therefore, discrimination will increase with trophic level as carnivores ingest more protein than herbivores and omnivores or (2) nitrogen discrimination increases as dietary protein quality decreases and, therefore, discrimination will decrease with trophic level as carnivores ingest higher quality protein than do herbivores. Discrimination factors were summarized for five major diet groupings and 21 different species of birds and mammals. Discrimination did not differ between mammals and birds and decreased as protein quality (expressed as biological value) increased with trophic level (i.e., herbivores to carnivores). Relationships between discrimination factors and dietary nitrogen concentration or C:N ratios were either the opposite of what was hypothesized or non-significant. Dietary protein quality accounted for 72% of the variation in discrimination factors across diet groupings. We concluded that protein quality established the baseline for discrimination between dietary groupings, while other variables, such as dietary protein intake relative to animal requirements, created within-group variation. We caution about the care needed in developing studies to understand variation in discrimination and subsequently applying those discrimination factors to estimate assimilated diets of wild animals.     

Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization

To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.     

Trophic structure of a coastal fish community determined with diet and stable isotope analyses

A combination of dietary guild analysis and nitrogen (δ¹⁵N) and carbon (δ¹³C) stable‐isotope analysis was used to assess the trophic structure of the fish community in Rhode Island and Block Island Sounds, an area off southern New England identified for offshore wind energy development. In the autumn of 2009, 2010 and 2011, stomach and tissue samples were taken from 20 fish and invertebrate species for analysis of diet composition and δ¹⁵N and δ¹³C signatures. The food chain in Rhode Island and Block Island Sounds comprises approximately four trophic levels within which the fish community is divided into distinct dietary guilds, including planktivores, benthivores, crustacivores and piscivores. Within these guilds, inter‐species isotopic and dietary overlap is high, suggesting that resource partitioning or competitive interactions play a major role in structuring the fish community. Carbon isotopes indicate that most fishes are supported by pelagic phytoplankton, although there is evidence that benthic production also plays a role, particularly for obligate benthivores such as skates Leucoraja spp. This type of analysis is useful for developing an ecosystem‐based approach to management, as it identifies species that act as direct links to basal resources as well as species groups that share trophic roles.     

Sources of organic matter and methylmercury in littoral macroinvertebrates: a stable isotope approach

The main objective of this study was to assess organic matter (OM) and methylmercury (MeHg) sources for freshwater littoral macroinvertebrate primary consumers. The carbon and nitrogen stable isotope ratios (δ¹³C, δ¹⁵N) of sources (epiphytes, macrophytes, suspended particulate matter _SPM) and of macroinvertebrate consumers were measured in a fluvial lake with extensive macrophyte beds (emergent and submerged). To determine the relative contribution of each OM source to macroinvertebrate diets we used the IsoSource model that examines all possible combinations of solutions for each source. Total and MeHg concentrations of consumers were also measured. Results show that epiphytes and macrophytes are dominant in the diet of macroinvertebrates, especially in early summer (July). In mid-summer (August), SPM constitutes a non-negligible OM source to the primary consumers. Hg concentrations were higher in epiphytes than in the other OM sources. The proportion of epiphytes in macroinvertebrate diet was positively correlated with the percentage of MeHg in their tissues. There was no relationship between SPM assimilation and Hg concentration in macroinvertebrate consumers. These results suggest that epiphytes and macrophytes constitute the main pathway of Hg bioaccumulation in littoral food webs.