Degradation of biomolecules in bones: effects of the biological forensics as an example of the stability of isotope ratios in collagen

Modern bone samples were experimentally degraded by incubation into water at increased temperature and examined in terms of their collagen content, the stable C and N isotopic ratios, and the molar C/N ratio. The same analyses were carried out with archaeological human bone of varying age (300 up to 8000 years). The experimentally degraded samples exhibited changes of the collagen's integrity, which influence the stable isotope ratios. In the case of the archaeological material, a correlation between stable delta13C- and delta15N-values and collagen content could be demonstrated. The molar C:N ratio was no suitable criterion for the assessment of the state of preservation of extractable collagen.     

Effects of preservation method on stable carbon and nitrogen isotope values

Some methods of tissue preservation have significant effects on values of stable isotopes of carbon (delta(13)C) and nitrogen (delta(15)N), but studies on this topic are scattered in the literature. The goals of this study were to (1) summarize the results from studies of preservation effects in the literature and (2) test the effects of four common preservatives on delta(13)C and delta(15)N in epidermis tissue of three turtle species. Turtle tissue samples were subjected to up to five time intervals in five methods of preservation: drying at 60 degrees C for 24 h (the control), immersion in a 70% ethanol solution, immersion in a saturated NaCl aqueous solution, freezing at -10 degrees C in a frost-free freezer, and immersion in a dimethyl sulfoxide (DMSO)-ethylenediaminetetraacetic acid buffer. The delta(13)C and delta(15)N values for tissues preserved in 70% ethanol and NaCl aqueous solution were not significantly different from those of tissues dried at 60 degrees C, but samples preserved in DMSO were significantly different from dried samples. Freezing preservation had a significant effect on delta(13)C and delta(15)N at 60 d, which may have resulted from the use of a frost-free freezer. The effects of 20 different preservative methods on delta(13)C and delta(15)N in different tissues are summarized.     

Stable nitrogen and carbon isotope ratios in bone collagen as indices of prehistoric dietary dependence on marine and terrestrial resources in southern California

The ratios of 15N to 14N and 13C to 12C tend to be higher in marine than in terrestrial organisms. The concentrations of these isotopes in human bone collagen consequently can be used to make inferences about the contribution of marine and terrestrial resources to prehistoric diets. The utility of studying 15N/14N and 13C/12C ratios in conjunction with each other is illustrated by our analysis of 40 human burials from archaeological sites in the Santa Barbara Channel area of southern California. The mean delta 13C and delta 15N values (in per mil) of collagen from these skeletons decrease progressively from the Channel Islands (delta 13C = -14.0, delta 15N = +16.3) to the mainland coast (delta 13C = -14.5, delta 15N = +14.9) to the interior (delta 13C = -17.2, delta 15N = +10.9). These data suggest that Indians living on the Channel Islands during the late prehistoric period were heavily dependent on marine resources. The inhabitants of the mainland interior, in contrast, had a diet composed largely of terrestrial foods. From their isotope ratios, it appears that the Indians who lived on the mainland coast consumed a mixed diet containing substantial quantities of both marine and terrestrial resources. Differences in 15N/14N and 13C/12C ratios of individuals from mainland sites dating from the early and late prehistoric periods show that the marine component of the diet increased substantially through time. These isotopic data are consistent with pathological, faunal, and artifactual evidence of increased marine resource exploitation during the late prehistoric period.     

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.     

Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

Stable isotope analysis of carbon (C) and nitrogen (N) in animal tissues is an important approach to investigate the tropic status and habitat of marine species. Some biases due to lipid extractions and preservation can hinder the interpretation of results, yet their effects have not been investigated in squid. In this study, we evaluated the effects of lipid extraction and preservatives (dimethylsulfoxide (DMSO), 70% ethanol, and 10% buffered formaldehyde) on the δ¹³C, δ¹⁵N and C:N ratios in squid muscle. Beaks were placed under the same treatments with the exception of DMSO. Muscle and beak samples remained under treatment for 375 days and 416 days, respectively. Our results indicate that lipid extractions increased the mean values of unpreserved samples by 0.8‰ for δ¹³C and by 0.68‰ for δ¹⁵N. Preservatives also affected the isotopic composition in muscle at different magnitudes. DMSO remarkably reduced and increased the variability for δ¹³C and δ¹⁵N values among samples, formalin mainly reduced δ¹³C values by 1.5‰, whereas ethanol increased both δ¹³C and δ¹⁵N by ≤ 0.8‰. Lipid extractions eliminated the effect of DMSO and ethanol for δ¹³C and δ¹⁵N, and formalin only for δ¹⁵N. In beak, negligible shifts in δ¹³C, δ¹⁵N and C:N ratios were recorded after preservation in ethanol and formalin. Although lipid extractions can be recommended to reduce the effect of preservation, further research is needed to develop correction models for isotopic shifts associated with both lipid extractions in unpreserved and preserved muscle tissues. Lipid extractions per se could introduce a bias that may have important implications for ecological studies.     

Using stable isotope analysis to obtain dietary profiles from old hair: a case study from Plains Indians

Stable isotope composition of human tissue reflects that of foods consumed, and can provide information about diet independent of artifactual remains. Here we refine and test this method by analyzing nitrogen (delta(15)N) and carbon (delta(13)C) isotope ratios in historic North American Plains Indians hair. Gas-source isotope-ratio mass spectrometry provides high-precision data for both delta(15)N and delta(13)C (+/-0.2 per thousand, 1 sigma) in single hair strands as short as 2 cm (100-150 mug). Because hair contains more carbon than nitrogen, if only delta(13)C data are needed, shorter strands (<1 cm) can be analyzed. This reduction in sample size opens new opportunities for analysis of small hair fragments found in archaeological excavations, as well as for analysis of seasonal variations in long hair strands. We find distinct isotope profiles (delta(15)N vs. delta(13)C) for two cultural groups, the Lower Brule reservation Sioux of 1892 and the reservation Blackfoot of 1892 and 1935. The resultant dietary profiles indicate a higher consumption of meat by the Blackfoot and a higher consumption of maize (or of animals that had fed on maize or other C(4) plants) by the Lower Brule. The two groups of Blackfoot yield similar isotopic profiles despite the passage of four decades, suggesting a strong role for cultural preference even as food sources change. Such stable isotope profiles can be used to link samples from the same cultural tradition based on their similar diets.     

Isotopic discrimination between food and blood and feathers of captive penguins: implications for dietary studies in the wild

Using measurements of naturally occurring stable isotopes to reconstruct diets or source of feeding requires quantifying isotopic discrimination factors or the relationships between isotope ratios in food and in consumer tissues. Diet-tissue discrimination factors of carbon ((13)C/(12)C, or delta (13)C) and nitrogen ((15)N/(14)N, or delta (15)N) isotopes in whole blood and feathers, representing noninvasive sampling techniques, were examined using three species of captive penguins (king Aptenodytes patagonicus, gentoo Pygoscelis papua, and rockhopper Eudyptes chrysocome penguins) fed known diets. King and rockhopper penguins raised on a constant diet of herring and capelin, respectively, had tissues enriched in (15)N compared to fish, with discrimination factors being higher in feathers than in blood. These data, together with previous works, allowed us to calculate average discrimination factors for (15)N between whole lipid-free prey and blood and feathers of piscivorous birds; they amount to +2.7 per thousand and +4.2 per thousand, respectively. Both fish species were segregated by their delta (13)C and delta (15)N values, and importantly, lipid-free fish muscle tissue was consistently depleted in (13)C and enriched in (15)N compared to whole lipid-free fish. This finding has important implications because previous studies usually base dietary reconstructions on muscle of prey rather than on whole prey items consumed by the predator. We tested the effect of these differences using mass balance calculations to the quantification of food sources of gentoo penguins that had a mixed diet. Modeling indicated correct estimates when using the isotopic signature of whole fish (muscle) and the discrimination factors between whole fish (muscle) and penguin blood. Conversely, the use of isotopic signatures of muscle together with discrimination factors between whole fish and blood (or the reverse) leads to spurious estimates in food proportions. Consequently, great care must be taken in the choice of isotopic discrimination factors to apply to wild species for which no controlled experiments on captive individuals have been done. Finally, our results also indicate that there is no need to remove lipids before isotopic analysis of avian blood.     

Nutrient routing in omnivorous animals tracked by stable carbon isotopes in tissue and exhaled breath

Omnivorous animals feed on several food items that often differ in macronutrient and isotopic composition. Macronutrients can be used for either metabolism or body tissue synthesis and, therefore, stable C isotope ratios of exhaled breath (delta(13)C(breath)) and tissue may differ. To study nutrient routing in omnivorous animals, we measured delta(13)C(breath) in 20-g Carollia perspicillata that either ate an isotopically homogeneous carbohydrate diet or an isotopically heterogeneous protein-carbohydrate mixture. The delta(13)C(breath) converged to the delta(13)C of the ingested carbohydrates irrespective of whether proteins had been added or not. On average, delta(13)C(breath) was depleted in (13)C by only ca. -2 per thousand in relation to the delta(13)C of the dietary carbohydrates and was enriched by +8.2 per thousand in relation to the dietary proteins, suggesting that C. perspicillata may have routed most ingested proteins to body synthesis and not to metabolism. We next compared the delta(13)C(breath) with that of wing tissue (delta(13)C(tissue)) in 12 free-ranging, mostly omnivorous phyllostomid bat species. We predicted that species with a more insect biased diet--as indicated by the N isotope ratio in wing membrane tissue (delta(15)N(tissue))--should have higher delta(13)C(tissue) than delta(13)C(breath) values, since we expected body tissue to stem mostly from insect proteins and exhaled CO(2) to stem from the combustion of fruit carbohydrates. Accordingly, delta(13)C(tissue) and delta(13)C(breath) should be more similar in species that feed predominantly on plant products. The species-specific differences between delta(13)C(tissue) and delta(13)C(breath) increased with increasing delta(15)N(tissue), i.e. species with a plant-dominated diet had similar delta(13)C(tissue) and delta(13)C(breath) values, whereas species feeding at a higher trophic level had higher delta(13)C(tissue) than delta(13)C(breath) values. Our study shows that delta(13)C(breath) reflect the isotope ratio of ingested carbohydrates, whereas delta(13)C of body tissue reflect the isotope ratio of ingested proteins, namely insects, supporting the idea of isotopic routing in omnivorous animals.     

Stable isotopes, beaks and predators: a new tool to study the trophic ecology of cephalopods, including giant and colossal squids

Cephalopods play a key role in the marine environment but knowledge of their feeding habits is limited by lack of data. Here, we have developed a new tool to investigate their feeding ecology by combining the use of their predators as biological samplers together with measurements of the stable isotopic signature of their beaks. Cephalopod beaks are chitinous hard structures that resist digestion and the stable isotope ratios of carbon (delta13C) and nitrogen (delta15N) are indicators of the foraging areas and trophic levels of consumers, respectively. First, a comparison of delta13C and delta15N values of different tissues from the same individuals showed that beaks were slightly enriched in 13C but highly impoverished in 15N compared with lipid-free muscle tissues. Second, beaks from the same species showed a progressive increase in their delta15N values with increasing size, which is in agreement with a dietary shift from lower to higher trophic levels during cephalopod growth. In the same way, there was an increase in the delta15N signature of various parts of the same lower beaks in the order rostrum, lateral walls and wings, which reflects the progressive growth and chitinization of the beaks in parallel with dietary changes. Third, we investigated the trophic structure of a cephalopod community for the first time. Values of delta15N indicate that cephalopods living in slope waters of the subantarctic Kerguelen Islands (n=18 species) encompass almost three distinct trophic levels, with a continuum of two levels between crustacean- and fish-eaters and a distinct higher trophic level occupied by the colossal squid Mesonychoteuthis hamiltoni. delta13C values demonstrated that cephalopods grow in three different marine ecosystems, with 16 species living and developing in Kerguelen waters and two species migrating from either Antarctica (Slosarczykovia circumantarctica) or the subtropics (the giant squid Architeuthis dux). The stable isotopic signature of beaks accumulated in predators' stomachs therefore revealed new trophic relationships and migration patterns and is a powerful tool to investigate the role of the poorly known cephalopods in the marine environment.     

Detection of breastfeeding and weaning in modern human infants with carbon and nitrogen stable isotope ratios

Carbon ((13)C/(12)C) and nitrogen ((15)N/(14)N) stable isotope ratios were longitudinally measured in fingernail and hair samples from mother-infant pairs where infants were exclusively breastfed (n = 5), breast- and formula-fed (n = 2), or exclusively formula-fed (n = 1) from birth. All exclusively breastfed infants had a dual enrichment in carbon ( approximately 1 per thousand) and nitrogen ( approximately 2-3 per thousand) when compared to maternal values. In contrast, breast- and formula-fed subjects had reduced enrichments compared to exclusively breastfed subjects, and the exclusively formula-fed infant showed no increase in delta(13)C or delta(15)N values. This finding of a carbon trophic level effect in breastfeeding infants suggests that (13)C-enrichments of approximately 1 per thousand in archaeological populations are not necessarily the result of the consumption of C(4)-based weaning foods such as maize or millet. During the weaning process, the delta(13)C results for breastfed infants declined to maternal levels more rapidly than the delta(15)N results. This suggests that delta(13)C values have the potential to track the introduction of solid foods into the diet, whereas delta(15)N values monitor the length of time of breast milk consumption. These findings can be used to refine the isotopic analysis of breastfeeding and weaning patterns in past and modern populations.     

Preservation effects on stable isotope ratios and consequences for the reconstruction of energetic pathways

Stable isotope analysis provides a powerful tool for describing the energetic pathways in a variety of ecosystems. However, isotope ratios of animal tissues can be altered by preservation methods, potentially leading to biased estimates of energy pathways when they are not taken into account. Here, we investigated the direct preservation effects of formalin, ethanol, NaCl, and drying on the δ¹³C and δ¹⁵N of fish muscle tissues, as well as the ultimate effects on the reconstruction of the energy pathways. All preservation methods, except drying, had significant impacts on δ¹³C and δ¹⁵N values. The effects of preservation appear to be highly taxa-specific and no significant time-dependent variations in nearly 2-year duration of preservation. δ¹³C and δ¹⁵N values were generally changed dramatically within the early stage of the preservation process and became stable over a relatively long-term preservation. Using an isotopic balance mixing model, the isotope-based food web reconstruction reveals that, without preservation correction, the importance of the pelagic energetic pathways for the fishes could be misestimated, except for the drying preservation. These results highlight that preservation can bias the interpretation of food web reconstruction results.     

Intraspecific variation in hair delta(13)C and delta(15)N values of ring-tailed lemurs (Lemur catta) with known individual histories, behavior, and feeding ecology

Stable carbon and nitrogen isotope compositions were analyzed from hair samples of 30 sympatric ring-tailed lemurs (Lemur catta) inhabiting the Beza Mahafaly Special Reserve, Madagascar. All lemurs were known individuals involved in a longitudinal study, which allowed us to explore the degree to which group membership, sex, health status, and migration influenced their stable isotope compositions. The differences in delta(13)C and delta(15)N values between groups were small (<1.5 per thousand) but highly significant. In fact, each group was tightly clustered, and discriminant function analysis of the stable isotope data assigned individuals to the group in which they were originally collared with over 90% accuracy. In general, the differences between groups reflected the degree to which they utilized forested versus open habitats. As open habitats at Beza Mahafaly often correspond to areas of anthropogenic disturbance, these data suggest that isotopic data can be useful for addressing questions of lemur conservation. There were few sex differences, but significant differences did occur between individuals of normal and suboptimal health, with those in poor health (especially those in the worst condition) being enriched in (15)N and to a lesser degree (13)C compared with healthy individuals. Moreover, lemurs that had emigrated between 2003 and 2004 had different delta(13)C and delta(15)N compositions than their original groups.     

Subsistence strategies of two "savanna" chimpanzee populations: the stable isotope evidence.

Twenty-two chimpanzee hair samples collected from night nests at two different "savanna" sites were analyzed for carbon and nitrogen stable isotope ratios represented as delta13C and delta15N values. The first at Ugalla, Tanzania is a miombo woodland with grass groundcover and small patches of forest. The second at Ishasha, Democratic Republic of the Congo is a habitat composed of riverine gallery forest, semideciduous thicket forest, wooded grassland, and grassland. Based on comparative data from other primates, Ugalla hair delta13C values suggest that the chimpanzees are feeding primarily in the woodland rather than in forest patches or on grassland foods (grasses or grammivorous fauna). Similar comparisons indicate that the Ishasha chimpanzees are feeding within the forests and not in more open areas. In addition, the Ugalla chimpanzees had delta15N values that indicate extensive ingestion of leguminous flowers, seeds, and/or leaves. The Ishasha samples show a range encompassing three trophic levels. Two samples with the most positive values may indicate a nursing signal or vertebrate-feeding. Three individuals with intermediate values are similar to those in omnivorous nonhuman primate species. The four individuals with the lowest values are very similar to those in herbivorous monkeys. Stable isotope ratios permit time-averaged and habitat-specific dietary comparisons among sites, even without habituation or detailed foraging observations.     

Stable isotopes document seasonal changes in trophic niches and winter foraging individual specialization in diving predators from the Southern Ocean

1. Climatic variation outside the breeding season affects fluctuations in population numbers of seabirds and marine mammals. A challenge in identifying the underlying biological mechanisms is the lack of information on their foraging strategies during winter, when individuals migrate far from their breeding grounds. 2. We investigated the temporal variability in resource partitioning within the guild of five sympatric Subantarctic penguins and fur seals from Crozet Islands. The stable isotopic ratios of carbon (delta(13)C) and nitrogen (delta(15)N) for whole blood were measured for penguins and fur seals, as were the isotopic ratios for penguin nails and food. Animals were sampled at two periods, during breeding in summer and at their arrival in the colonies in spring (hereafter winter, since the temporal integration of blood amounting to several months). 3. In summer, delta(13)C and delta(15)N for blood samples defined three foraging areas and two trophic levels, respectively, characterizing four nonoverlapping trophic niches. King penguins and female Antarctic and Subantarctic fur seals are myctophid eaters foraging in distinct water masses, while both macaroni and rockhopper penguins had identical isotopic signatures indicating feeding on crustaceans near the archipelago. 4. Isotopic ratios were almost identical in summer and winter suggesting no major changes in the species niches, and hence, in the trophic structure of the guild during the nonbreeding period. A seasonal difference, however, was the larger variances in delta(13)C (and also to a lesser extent in delta(15)N) values in winter, thus verifying our hypothesis that trophic niches widen when individuals are no longer central place foragers. 5. Winter isotopic ratios of macaroni penguins and male Antarctic fur seals had large variances, indicating individual foraging specializations. The range of delta(13)C and delta(15)N values of male fur seals showed, respectively, that they dispersed over a wide latitudinal gradient (from Antarctica to north of the archipelago) and fed on different prey (crustaceans and fish). 6. By comparing summer and winter isotopic ratios and examining the summer diet, we highlight the feeding habits of marine predators that were not previously addressed. The findings have a number of implications for understanding the functioning of the pelagic ecosystem and on the demography of these species.     

Annual rainfall does not directly determine the carbon isotope ratio of leaves of Eucalyptus species

Leaf carbon isotope discrimination (delta13C) was widely considered to directly reflect the rainfall environment in which the leaf developed, but recent observations have queried this. The relationship between delta13C and rainfall was explored in Eucalyptus species growing along a rainfall gradient in Australia. The leaves of 43 species of Eucalyptus and the closely related Corymbia species produced in 2003 were sampled in September 2004 at 50 sites and grouped into 15 locations along a rainfall gradient in southwest Western Australia. At 24 sites, the same species and same trees were sampled as in a study in September 2003 when leaves produced in 2002 were sampled. The rainfall in 2004 was on average 190 mm (range 135-270 mm) higher at all locations than in 2003. In the leaves sampled in 2004, the mean carbon isotope discrimination (delta13C) across the 15 locations decreased 2.9 per thousand per 1000 mm of rainfall, the specific leaf area (SLA) increased by 2.9 m2 kg(-1) per 1000 mm of rainfall and the nitrogen (N) content decreased by 1.56 g m(-2) per 1000 mm of rainfall. In contrast, a comparison between the leaves produced in the drier 2002 year compared with the wetter 2003 year showed that there was a strong correlation (r2= 0.85) between the SLA values between years and a trend for higher values with increasing SLA, but the values of delta(13)C were on average only 0.38 per thousand lower (more negative) at all locations in the wetter year, equivalent to a decrease of 2.0 per thousand per 1000 mm of rainfall. The results suggest that while there may be constitutive differences in leaf morphology, SLA and N content per unit area, increasing rainfall or cloudiness associated with higher rainfall increases SLA and decreases N content per unit area. We conclude that rainfall does not directly influence delta13C, but induces leaf morphological and physiological changes that affect the resultant delta13C.     

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird

The use of stable isotopes to infer diet requires quantifying the relationship between diet and tissues and, in particular, knowing of how quickly isotopes turnover in different tissues and how isotopic concentrations of different food components change (discriminate) when incorporated into consumer tissues. We used feeding trials with wild-caught yellow-rumped warblers (Dendroica coronata) to determine delta15N and delta13C turnover rates for blood, delta15N and delta13C diet-tissue discrimination factors, and diet-tissue relationships for blood and feathers. After 3 weeks on a common diet, 36 warblers were assigned to one of four diets differing in the relative proportion of fruit and insects. Plasma half-life estimates ranged from 0.4 to 0.7 days for delta13C and from 0.5 to 1.7 days for delta15N . Half-life did not differ among diets. Whole blood half-life for delta13C ranged from 3.9 to 6.1 days. Yellow-rumped warbler tissues were enriched relative to diet by 1.7-3.6% for nitrogen isotopes and by -1.2 to 4.3% for carbon isotopes, depending on tissue and diet. Consistent with previous studies, feathers were the most enriched and whole blood and plasma were the least enriched or, in the case of carbon, slightly depleted relative to diet. In general, tissues were more enriched relative to diet for birds on diets with high percentages of insects. For all tissues, carbon and nitrogen isotope discrimination factors increased with carbon and nitrogen concentrations of diets. The isotopic signature of plasma increased linearly with the sum of the isotopic signature of the diet and the discrimination factor. Because the isotopic signature of tissues depends on both elemental concentration and isotopic signature of the diet, attempts to reconstruct diet from stable isotope signatures require use of mixing models that incorporate elemental concentration.     

Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes

Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.     

Parasite-induced changes in nitrogen isotope signatures of host tissues

To estimate isotopic changes caused by trematode parasites within a host, we investigated changes in the carbon and nitrogen isotope ratios of the freshwater snail Lymnaea stagnalis infected by trematode larvae. We measured carbon and nitrogen stable isotopes within the foot, gonad, and hepatopancreas of both infected and uninfected snails. There was no significant difference in the delta13C and delta15N values of foot and gonad between infected and uninfected snails; thus, trematode parasite infections may not cause changes in snail diets. However, in the hepatopancreas, delta15N values were significantly higher in infected than in uninfected snails. The 15N enrichment in the hepatopancreas of infected snails is caused by the higher 15N ratio in parasite tissues. Using an isotope-mixing model, we roughly estimated that the parasites in the hepatopancreas represented from 0.8 to 3.4% of the total snail biomass, including the shell.     

Preservation Methods Alter Carbon and Nitrogen Stable Isotope Values in Crickets (Orthoptera: Grylloidea)

Stable isotope analysis (SIA) is an important tool for investigation of animal dietary habits for determination of feeding niche. Ideally, fresh samples should be used for isotopic analysis, but logistics frequently demands preservation of organisms for analysis at a later time. The goal of this study was to establish the best methodology for preserving forest litter-dwelling crickets for later SIA analysis without altering results. We collected two cricket species, Phoremia sp. and Mellopsis doucasae, from which we prepared 70 samples per species, divided among seven treatments: (i) freshly processed (control); preserved in fuel ethanol for (ii) 15 and (iii) 60 days; preserved in commercial ethanol for (iv) 15 and (v) 60 days; fresh material frozen for (vi) 15 and (vii) 60 days. After oven drying, samples were analyzed for δ ¹⁵N, δ ¹³C values, N(%), C(%) and C/N atomic values using continuous flow isotope ratio mass spectrometry. All preservation methods tested, significantly impacted δ ¹³C and δ ¹⁵N and C/N atomic values. Chemical preservatives caused δ ¹³C enrichment as great as 1.5‰, and δ ¹⁵N enrichment as great as 0.9‰; the one exception was M. doucasae stored in ethanol for 15 days, which had δ ¹⁵N depletion up to 1.8‰. Freezing depleted δ ¹³C and δ ¹⁵N by up to 0.7 and 2.2‰, respectively. C/N atomic values decreased when stored in ethanol, and increased when frozen for 60 days for both cricket species. Our results indicate that all preservation methods tested in this study altered at least one of the tested isotope values when compared to fresh material (controls). We conclude that only freshly processed material provides adequate SIA results for litter-dwelling crickets.     

A non‐lethal approach to estimate whole‐body 13C and 15N stable isotope ratios of freshwater amphipods using walking legs

To analyze the stable isotope ratios of small‐bodied invertebrates, the entire animal is typically sacrificed and processed, which is problematic for threatened or endangered species. Appendages which are regenerated could be used to infer whole‐body isotope ratios, but differences in turnover rates and isotopic signatures among tissues may confound such an approach. We tested the hypothesis that the δ¹³C and δ¹⁵N of whole‐body tissue for freshwater amphipods could be predicted from the δ¹³C and δ¹⁵N of walking legs, with the goal of estimating body δ¹³C and δ¹⁵N of Gammarus acherondytes, a United States federally endangered species. To test this, we analyzed the δ¹³C and δ¹⁵N of walking legs and bodies of five species of amphipods from geographically distant areas (Idaho, Illinois, and Washington) in the United States. The general relationships of whole‐body isotope ratios of C and N as a function of leg isotope ratios were linear and had slopes of one. In the range of the data, leg δ¹³C was slightly lower than body δ¹³C, indicating some tissue‐specific fractionation, while δ¹⁵N was similar for legs and bodies. Our data suggest that legs can be used to predict body isotope ratios in freshwater amphipods. This approach provides an additional tool to help researchers understand the biology of small, endangered invertebrates without sacrificing individuals. This is especially useful in cave ecosystems where populations are naturally sparse.