1. Stable isotope analyses coupled with mixing models are being used increasingly to evaluate ecological management issues and questions. Such applications of stable isotope analyses often require simultaneous carbon and nitrogen analyses from the same sample. Correction of the carbon isotope values to take account of the varying content of 13 C-depleted lipids is then frequently achieved by a lipid-normalization procedure using a model describing the relationship between change in δ 13 C following lipid removal and the original C:N ratio of a sample. 2. We evaluated the applicability of two widely used normalization models using empirical data for muscle tissue from a wide range of fish and for aquatic invertebrates. Neither normalization model proved satisfactory, and we present some modifications that greatly improve the fit of one of the models to the fish muscle data. For invertebrates we found no clear relationship between change in δ 13 C following lipid removal and the original C:N ratio. 3. We also examined the effect of lipid-normalization on the output of a mixing model designed to calculate the proportional contribution of prey items to the diet of a consumer. Mixing model output was greatly influenced by whether prey or consumer values alone or together were lipid-normalized and we urge caution in the interpretation of results from these models pending further experimental evidence. 4. Synthesis and applications . We describe a revised lipid-normalization model that should be applicable to a wide range of marine and freshwater fish species in studies applying stable isotope analyses to ecological management issues. However, we strongly advise against applying these kinds of lipid-normalization models to aquatic invertebrate data. The interpretation of outputs from mixing models is greatly influenced by whether the carbon isotope data have been lipid-normalized or not.

A revised model for lipid‐normalizing δ13C values from aquatic organisms, with implications for isotope mixing models

Summary
1Insects are frequently sampled in food web studies and are assumed to follow the pattern of enrichment in 15N across trophic levels observed for other organisms. However, few studies have examined 15N discrimination of metamorphosing insects.
2We measured the δ15N of larvae, pupae and adults of four Lepidoptera (Bombyx mori, Galleria mellonella, Manduca sexta and Vanessa cardui), one Diptera (Sarcophaga sp.) and one Coleoptera (Tenebrio molitor) fed on artificial diets.
3The tissues of larvae were enriched in 15N relative to their diet and this enrichment was explained by the production of 15N-depleted frass. Surprisingly, the tissues of adults were enriched in 15N relative to larvae in all but one species (T. molitor). Because, we measured the tissues of adults immediately after they emerged from pupal cases, the enrichment was not due to a change in diet. Rather, it was due to the excretion of 15N depleted metabolic waste (‘meconium’) that resulted from protein metabolism during metamorphosis.
4To our knowledge, the 15N enrichment that seems to accompany metamorphosis in holometabolous insects had not been previously recognized. This enrichment must be accounted for in ecological studies that rely on stable isotopes to identify both the trophic position and diet of insects.

Isotopic enrichment without change in diet: an ontogenetic shift in δ15N during insect metamorphosis

Stable oxygen and hydrogen isotope analyses of fossil aquatic organisms, such as the chitinous head capsules of chironomid larvae (Chironomidae: Diptera), are promising proxies for inferring paleoecological conditions. In order for analyses of stable oxygen (δ18O) and hydrogen isotope ratios (δ2H) of fossil chironomid head capsules to be used effectively in paleoecological research, it is necessary to understand the factors controlling their stable oxygen and hydrogen composition. We cultured chironomid larvae in two isotopically distinct waters under controlled, replicated laboratory conditions. Chironomid larvae were fed on identical diets, to examine the degree to which water and diet influence the δ18O and δ2H of these organisms. We used a two-end member mixing model to determine the proportional contributions of oxygen and hydrogen from water to the oxygen and hydrogen of chironomid larvae. Our experiment demonstrated that 69.0 ± 0.4% of oxygen and 30.8 ± 2.6% of hydrogen in chironomid larvae are derived from habitat water. Our results show that oxygen isotopes from chironomid remains can better constrain past habitat water isotopic changes compared to hydrogen, due to 69% of the chironomid oxygen being influenced by habitat water. Our data add to a small but growing suite of comparative data on the sources of oxygen and hydrogen in animal tissues, and provide the first such analyses from aquatic insects.

The influence of diet and water on the stable oxygen and hydrogen isotope composition of Chironomidae (Diptera) with paleoecological implications

Stable isotopic studies on chitin. III. The D/H and 18O/16O ratios in arthropod chitin

This study has been supported partially by funds of a bilateral programme between the CNR (Italy) and the CSIC (Spain), and partially by the EU-INCO project WADI (CT2005-15226)

Acid washing effect on elemental and isotopic composition of whole beach arthropods: Implications for food web studies using stable isotopes

Measurements of the stable isotope ratios 13C/12C and 15N/14N in chitin and chitosan isolates show large variations which reflect intrinsic compositional and isotopic heterogeneities as well as differences caused by methods of preparation. Analysis of D-glucosamine hydrochloride from chitin hydrolysates eliminates these problems.

M. J. DeNiro

Papers

Stable Isotopic Studies on Chitin, Measurements on Chitin/Chitosan Isolates and D-Glucosamine Hydrochloride from Chitin