Stable Isotope Analysis

Up until the mid 1980’s, the conventional method for determining the diet of marine or other reclusive predators, was to examine stomach contents, either by inducing vomiting or by necropsy (Hyslop, 1980). However, there are several biases to these methods including differential digestion rates, secondary digestion and differential energy contributions (Kelly 2000; Pearson

et al., 2003; Iverson et al., 2004; Iverson et al., 2007). In addition, stomach contents generally only reflect the last meal eaten and in the case of a dead animal, may not be representative of their normal diet e.g. if the animal had been sick (MacLeod et al, 2003). Technological advances now offer several alternative methodologies that replace, or complement stomach content analysis (Peterson & Fry, 1987, Kelly, 2000). One such biogeochemical technique is stable isotope analysis (Fisk et al., 2001; Cherel et al., 2005b). Isotopes are variations on the common atom structures found in biotic and abiotic materials. A ratio of the heavier isotope to the usually more common, lighter isotope in a sample is compared to international standards. In the context of analysing marine predator diets, the isotopic ratios of nitrogen, delta 15N (δ15N = 15N / 14N) and carbon, delta 13C (δ 13C = 13 C / 12C), are most commonly used and can be detected from almost any tissue e.g. feathers, blood, hair, nails, skin, all of which can be collected relatively non-invasively (Cherel et al., 2005a; Cherel & Hobson, 2007). Both ratios are expressed as parts per thousand (‰), and the international standards are Pee Dee Belemnite for carbon and atmospheric air for nitrogen. In an ecological context, the main principals assumed are: 1) that the isotopes in an animals tissues accurately reflect their diet; 2) as consumer tissues turnover at

81 different rates they can reflect diet on temporal or spatial scales (Bearhop et al., 2002), and 3) isotopes accumulate through the food web in a predictive manner, known as enrichment or fractionation (Cherel et al., 2005a).

4.2.1.1 Nitrogen

The isotope 15N is known to bio-accumulate in animal tissues because 14N is excluded preferentially in metabolic processes (Macko et al., 1986; Peterson & Fry, 1987; Kelly, 2000). Thus, animals that feed at higher trophic levels subsequently have higher δ15

N (Vanderklift & Ponsard, 2003; Cherel & Hobson, 2007). The difference of δ15

N between marine trophic levels, referred to as isotopic discrimination (Cherel et al., 2005a), is relatively consistent and widely accepted to be between 3-4‰ (Hobson et al., 1994; Post, 2002; Geurts, 2006). While trophic level does not identify specific prey, differentiation can be made for example, between a diet of vertebrates over invertebrates, and even between the relative sizes of prey. Stable isotope analysis (using muscle tissue) was successfully used by Peggy Ostrom (1993) to place the trophic level of Sowerby’s beaked whales (Mesoplodon bidens) as consumers of small squid in offshore waters. The findings provided additional evidence and confirmed speculation of the diet, made from previous observations and examination of stomach contents. Hawke & Holdaway (2009) used stable isotope analysis on the feathers of Bellbirds (Anthornis melanura) and Red-crowned Parakeets (Cyanoramphus movaezelandiae) to highlight the importance of petrel (Procellariiformes spp.) colonies in enriching nutrient-depleted forest soils.

4.2.1.2 Carbon

Terrestrial C3 and C4 plants have distinct carbon isotopic signatures because

of their different photosynthetic pathways (Kelly, 2000; Post, 2002), with marine C3 plants having an intermediate signature. δ13C does not tend to

accumulate up the food chain (trophic fractionation ~0-1‰), but instead can indicate the primary source of carbon (i.e. different foraging habitats) within the food web (Kelly, 2000; Post, 2002). This is because phytoplankton has

82 lighter δ13

C values than most inshore marine plants, making it possible to distinguish between inshore and pelagic (offshore) carbon sources (Hobson et al., 1994; Kelly, 2000; Cherel & Hobson, 2007). Marine studies have also shown that δ13

C is negatively correlated to latitude (Cherel & Hobson, 2007).

Typically lipids are depleted in δ13

C relative to other tissues, and because the amount of lipid species’ can have varies considerably, it is recommended as best practice to remove lipids from (most) tissues being sampled, prior to stable isotope analysis. This ensures the results are standardised and not influenced by the abundance or distribution of lipids associated with the tissue (Hobson & Clark, 1992; Kelly, 2000; Becker et al., 2007).

4.2.1.3 Stable isotopes as bio-indicators

Within an environment, isotopic ratios can be very sensitive to changes in ecological processes (Dawson & Siegwolf, 2007). Thus monitoring stable isotopes, along with other measurements, can potentially serve as both an early-warning system of major changes and a means of quantifying anthropogenic activity within ecosystems (Hobson et al., 1994; Williams et al.,

2007; Hobson, 2007). For example, in California, stable isotope analysis of feather samples from the endangered Marbled Murrelet (Brachyramphus marmoratus) compared the trophic level of modern birds with those from museum specimens up to 100 years old (Becker & Beissinger, 2005). It was found over this period that decreased prey resources (resulting from overfishing) had caused the endangered Murrelets to forage further down the food web on energetically inferior prey, which may have contributed to poorer reproduction and their subsequent listing under the US Endangered Species Act. In another study, stable isotope analysis on tissue samples from a variety of species in the Gulf of the Farallones food web, found significant correlation between the stable isotope of nitrogen (indicative of trophic level) and organochlorine contaminants (Jarman et al., 1996).

In assessing diet, stable isotopes are assimilated into the various animal tissues at different rates and thus have the advantage of being able to indicate diet over different temporal scales (Hobson et al., 1994; Cherel et al.,

83 2005b). I.e. blood reflects the diet of the previous days/weeks, hair up to 6 months, while bone integrates the diet over much longer periods, perhaps an entire lifetime (O’Connell & Hedges, 2001; Cherel et al., 2005b; Hobson, 2007). Today, stable isotope analysis is extensively used in studies of avian and mammalian trophic ecology, both terrestrial and marine, and is acknowledged as an important tool for ecologists (Kelly, 2000; Dawson & Siegwolf, 2007).

4.2.1.4 Discrimination

Stable isotope experiments using captive animals fed a known diet have highlighted that discrimination factors can vary considerably between species and even between tissues (fractionation), as different metabolic pathways assimilate isotopes at different rates (Gannes et al., 1997). Thus, results of stable isotope analysis undertaken on wild animals are difficult to interpret unless the specific discrimination factors are known. Cherel et al. (2005a) found significant isotopic differences between species of King Penguins (Aptenodytes patagonicus) fed solely on herring (Clupea harengus), Rockhopper Penguins (Eudyptes chrysocome) fed on capelin (Mallotus villosus) and Gentoo Penguins (Pygoscelis papua) fed on a mixed diet of both fish species. They also found differences within species between the different tissues of whole blood and feathers. Similarly, Hobson et al. (1996) were able to identify different discrimination factors between diet (herring) and the tissues of nail, hair, skin, whiskers and blood of captive harp seals (Pagophilus groenlandicus), harbour seals (Phoca vitulina), and ringed seals (Phoca hispida). Typically, discrimination factors fall between 0-2‰ for δ13C and 2-5‰ for δ15

N (Peterson & Fry, 1987; Kelly, 2000) and if unknown average discrimination values are generally accepted. To date, it is understood that a captive feeding trial for LP has not been previously undertaken.

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