A simple method for measuring carbon-13 fatty acid enrichment in the major lipid classes of microalgae using GC-MS

A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of13C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of13C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae.

Tracking spatial distribution of human-derived wastewater from Davis Station, East Antarctica, using δ15N and δ13C stable isotopes.

Stable isotope ratios, δ15N and δ13C were effectively used to determine the geographical dispersion of human derived sewage from Davis Station, East Antarctica, using Antarctic rock cod (Trematomus bernacchii). Fish within 0-4km downstream of the outfall exhibited higher δ15N and δ13C values relative to reference sites. Nitrogen in particular showed a stepped decrease in δ15N with increasing distance from the discharge point by 1-2‰. Stable isotopes were better able to detect the extent of wastewater contamination than other techniques including faecal coliform and sterol measures. Uptake and assimilation of δ15N and δ13C up to 4km from the outfall adds to growing evidence indicating the current level of wastewater treatment at Davis Station is not sufficient to avoid impact to the surrounding environment. Isotopic assimilation in T. bernacchii is a viable biomarker for investigation of initial sewage exposure and longer term monitoring in the future.

The relationship between carbon stable isotope ratios of hatchling down and egg yolk in Black-headed Gulls

We reconstructed the nutrient source for egg synthesis by sampling Black-headed Gull (Larus ridibundus) eggs for yolk, analyzing their carbon stable isotope ratio, and comparing that to hatchling down. Most of the variation in carbon stable isotope ratio was explained by differences between nests, the within-nest variation being explained by laying order. These data indicate significant differences in diet choice between individual females and changes in food choice or body-store dynamics during egg laying. Among-egg variation in the carbon stable isotope ratios of the yolk were closely reflected in the hatchling down, on average down being enriched by 3.1‰ δ13C relative to yolk. Our findings support the contention that pre-laying female diets can be evaluated from hatchling down.

Strategies for extending metabolomics studies with stable isotope labelling and fluxomics

This is a perspective from the peer session on stable isotope labelling and fluxomics at the Australian & New Zealand Metabolomics Conference (ANZMET) held from 30 March to 1 April 2016 at La Trobe University, Melbourne, Australia. This report summarizes the key points raised in the peer session which focused on the advantages of using stable isotopes in modern metabolomics and the challenges in conducting flux analyses. The session highlighted the utility of stable isotope labelling in generating reference standards for metabolite identification, absolute quantification, and in the measurement of the dynamic activity of metabolic pathways. The advantages and disadvantages of different approaches of fluxomics analyses including flux balance analysis, metabolic flux analysis and kinetic flux profiling were also discussed along with the use of stable isotope labelling in in vivo dynamic metabolomics. A number of crucial technical considerations for designing experiments and analyzing data with stable isotope labelling were discussed which included replication, instrumentation, methods of labelling, tracer dilution and data analysis. This report reflects the current viewpoint on the use of stable isotope labelling in metabolomics experiments, identifying it as a great tool with the potential to improve biological interpretation of metabolomics data in a number of ways.

Terrestrial detritus supports the food webs in lowland intermittent streams of south-eastern Australia : a stable isotope study

1. Large amounts of terrestrial detritus enter many low-order forested streams, and this organic
material is often the major basal resource in the metazoan food webs of such systems. However,
despite their apparently low biomass, algae are the dominant food of organisms in a number of
aquatic communities which conventionally would have been presumed to be dependent on
allochthonous detritus, particularly those in the tropics and also in lowland intermittent streams
in arid Australia.
2. The dual stable isotope signatures (d13C and d15N) of potential primary food sources were
compared with the isotopic signatures of common aquatic animals in lowland intermittent
streams in south-eastern Australia, in both spring and summer, to determine whether
allochthonous detritus was an important nutritional resource in these systems. The isotopic
signatures of the major potential allochthonous plant food sources (Eucalyptus, Phalaris and
Juncus) overlapped, but were distinct from algae and the dominant macrophytes growing in the
study reaches. The isotopic signatures of biofilm were more spatially and temporally variable
than those of the other basal resources.
3. Despite allochthonous detritus having relatively high C : N ratios compared to other
potential basal resources, results from ISOSOURCE mixing model calculations demonstrated
that this detritus, and the associated biofilm, were the major energy sources assimilated by
macroinvertebrate primary consumers in both spring and summer. The importance of these
energy sources was also reflected in animals higher in the food web, including predatory
macroinvertebrates and fish. These resources were supplemented by autochthonous sources of
higher nutritional value (i.e. filamentous algae and macrophytes, which had relatively low
C : N ratios) when they became more prolific as the streams dried to disconnected pools in
summer.
4. The results highlight the importance of allochthonous detritus (particularly from Eucalyptus)
as a dependable energy source for benthic macroinvertebrates and fish in lowland intermittent
streams of south-eastern Australia. This contrasts with previous stable isotope studies
conducted in lowland intermittent streams in arid Australia, which have reported that the fauna
are primarily dependent on autochthonous algae.

High individual repeatability and population differentiation in stable isotope ratios in winter-grown collared flycatcher Ficedula albicollis feathers

For migrants, we often lack complete information of their spatial distribution year round. Here, we used stable carbon, nitrogen and hydrogen isotope ratios extracted from feathers grown at the wintering sites of the long-distance migratory collared flycatcher Ficedula albicollis, to study how individuals from different breeding populations are distributed at the wintering sites. A sub-sample of birds was also sampled in two consecutive years to test for the repeatability of isotope ratios. Birds from the same breeding populations had more similar isotope ratios compared to birds from other nearby populations (10–100 km apart). Furthermore, isotope repeatability within individuals was high, implying that the observed pattern of isotope variation is consistent between years. We put forward two hypotheses for these patterns; 1) strong wintering site philopatry and migratory connectivity, suggesting that migratory connectivity may potentially be found on a much smaller spatial scale than previously considered, and 2) consistent interpopulation differentiation of feeding ecology at their wintering site.

Trophic effects of fishing southern rock lobster Jasus edwardsii shown by combined fatty acid and stable isotope analyses

The southern rock lobster Jasus edwardsii is a commercial species that has benefited from the complete protection offered by no-take reserves, with higher abundances and larger animals recorded in reserves than in adjacent fished areas. What remains unclear is whether there is any change in the diet of lobsters in reserves, for example, as a result of increased intraspecific competition for food. We used combined chemical tracers to examine the diet of lobsters in fished and reserve areas in 2 bioregions in eastern Tasmania. δ15N values of lobsters were richer in fished than in reserve areas, indicating that lobsters eat a greater proportion of food items from higher trophic levels in fished areas. Mixing models suggest that ascidians, sea urchins and the turbinid gastropod were all important food sources for lobsters, but the importance of these food items differed between bioregions. This spatial variability may suggest that the small size of the reserve in one bioregion is inadequate at ensuring the diet of lobsters is protected from fishing pressure. Fatty acid profiles of lobsters supported the importance of these food sources to lobsters. Differences between bioregions, or inside and outside of reserves, were not apparent using fatty acids. The present study highlights that lobster fishing has the capacity to alter the trophic status of prey for generalist predators and suggests that fatty acid analyses may be limited in detecting changes in the dietary composition of such generalist feeders.

Stable isotope-based community metrics as a tool to identify patterns in food web structure in east African estuaries

1.Quantitative tools to describe biological communities are important for conservation and ecological management. The analysis of trophic structure can be used to quantitatively describe communities. Stable isotope analysis is useful to describe trophic organization, but statistical models that allow the identification of general patterns and comparisons between systems/sampling periods have only recently been developed. 2.Here, stable isotope-based Bayesian community-wide metrics are used to investigate patterns in trophic structure in five estuaries that differ in size, sediment yield and catchment vegetation cover (C3/C4): the Zambezi in Mozambique, the Tana in Kenya and the Rianila, the Betsiboka and Pangalanes Canal (sampled at Ambila) in Madagascar. 3.Primary producers, invertebrates and fish of different trophic ecologies were sampled at each estuary before and after the 2010–2011 wet season. Trophic length, estimated based on δ15N, varied between 3·6 (Ambila) and 4·7 levels (Zambezi) and did not vary seasonally for any estuary. Trophic structure differed the most at Ambila, where trophic diversity and trophic redundancy were lower than at the other estuaries. Among the four open estuaries, the Betsiboka and Tana (C4-dominated) had lower trophic diversity than the Zambezi and Rianila (C3-dominated), probably due to the high loads of suspended sediment, which limited the availability of aquatic sources. 4.There was seasonality in trophic structure at Ambila and Betsiboka, as trophic diversity increased and trophic redundancy decreased from the prewet to the postwet season. For Ambila, this probably resulted from the higher variability and availability of sources after the wet season, which allowed diets to diversify. For the Betsiboka, where aquatic productivity is low, this was likely due to a greater input of terrestrial material during the wet season. 5.The comparative analysis of community-wide metrics was useful to detect patterns in trophic structure and identify differences/similarities in trophic organization related to environmental conditions. However, more widespread application of these approaches across different faunal communities in contrasting ecosystems is required to allow identification of robust large-scale patterns in trophic structure. The approach used here may also find application in comparing food web organization before and after impacts or monitoring ecological recovery after rehabilitation.

Effects of diet change on carbon and nitrogen stable-isotope ratios in blood cells and plasma of the long-nosed bandicoot (Perameles nasuta)

Carbon (C) and nitrogen (N) stable isotopes offer a powerful tool for assessing the extent of tissue assimilation of dietary components. However, the method relies on knowledge of diet-tissue isotopic discrimination and how quickly diet shifts become apparent in various tissues. In the present study, blood plasma and blood cells, tissues that are easily obtained under field conditions, were used to validate the stable isotope method over a period of 4-5 weeks using captive long-nosed bandicoots (Perameles nasuta). Diet-tissue discrimination effects appeared to be small. For C, derived diet-tissue isotopic discriminations were 1.4‰ for blood plasma and -0.2‰ for blood cells. For N the values were 2.8‰ and 2.1‰, respectively, and were independent of the nitrogen content of the food. C and N turnover measurements in the blood plasma and cells of the bandicoots indicated that blood plasma provides dietary information integrated over a period of ∼3 weeks, whereas blood cells give an impression of the assimilated diet over a period of as much as half a year. These turnover rates were low compared with the little information available for birds and eutherian mammals, and probably relate to the typically low metabolic rate of marsupials.