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1. Dietary protein content and digestibility influences discrimination of amino acid nitrogen isotope values in a terrestrial omnivorous mammal

   https://doi.org/10.1002/rcm.9073  

   Whiteman, John P. ; Rodriguez Curras, Mauriel ; Feeser, Kelli L. ; Newsome, Seth D. ( April 2021 , Rapid Communications in Mass Spectrometry)

   Ecologists increasingly determine the δ¹⁵N values of amino acids (AA) in animal tissue; “source” AA typically exhibit minor variation between diet and consumer, while “trophic” AA have increased δ¹⁵N values in consumers. Thus, trophic‐source δ¹⁵N offsets (i.e., Δ¹⁵N_T‐S) reflect trophic position in a food web. However, even minor variations in δ¹⁵N_{source AA}values may influence the magnitude of offset that represents a trophic step, known as the trophic discrimination factor (i.e., TDF_T‐S). Diet digestibility and protein content can influence the δ¹⁵N values of bulk animal tissue, but the effects of these factors on AA Δ¹⁵N_T‐Sand TDF_T‐Sin mammals are unknown.

   We fed captive mice (Mus musculus) either (A) a low‐fat, high‐fiber diet with low, intermediate, or high protein; or (B) a high‐fat, low‐fiber diet with low or intermediate protein. Mouse muscle and dietary protein were analyzed for bulk tissue δ¹⁵N using elemental analyzer‐isotope ratio mass spectrometry (EA‐IRMS), and were also hydrolyzed into free AA that were analyzed for δ¹⁵N using gas chromatography‐combustion‐IRMS.

   As dietary protein increased, Δ¹⁵N_{Consumer‐Diet}slightly declined for bulk muscle tissue in both experiments; increased for AA in the low‐fat, high‐fiber diet (A); and remained the same or decreased for AA in the high‐fat, low‐fiber diet (B). The effects of dietary protein on Δ¹⁵N_T‐Sand on TDF_T‐Svaried by AA but were consistent between variables.

   Diets were less digestible and included more protein in Experiment A than in Experiment B. As a result, the mice in Experiment A probably oxidized more AA, resulting in greater Δ¹⁵N_{Consumer‐Diet}values. However, the similar responses of Δ¹⁵N_T‐Sand of TDF_T‐Sto diet variation suggest that if diet samples are available, Δ¹⁵N_T‐Saccurately tracks trophic position. If diet samples are not available, the patterns presented here provide a basis to interpret Δ¹⁵N_T‐Svalues. The trophic‐source offset of Pro‐Lys did not vary across diets, and therefore may be more reliable for omnivores than other offsets (e.g., Glu‐Phe).

    

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2. Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry

   https://doi.org/10.1002/rcm.9007  

   Phillips, Alexandra A. ; Wu, Fenfang ; Sessions, Alex L. ( January 2021 , Rapid Communications in Mass Spectrometry)

   Sulfur isotope analysis of organic sulfur‐containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural‐abundance sulfur isotopic compositions of the sulfur‐containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields.

   Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed‐phase high‐performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ³⁴S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature‐ramped chromatographic column and programmable helium carrier flow rates.

   On‐column focusing of SO₂in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1–0.3‰ 1 s.d.) δ³⁴S measurements of 1 to 10 μg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof‐of‐concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ³⁴S values of cysteine and methionine that can be connected to biosynthetic pathways.

   We have developed a sensitive, precise method for measuring the natural‐abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis.

    

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3. Carbon and nitrogen isotope fractionation of amino acids in an avian marine predator, the gentoo penguin ( Pygoscelis papua )

   https://doi.org/10.1002/ece3.1437  

   McMahon, Kelton W. ; Polito, Michael J. ; Abel, Stephanie ; McCarthy, Matthew D. ; Thorrold, Simon R. ( February 2015 , Ecology and Evolution)

   Compound‐specific stable isotope analysis (CSIA) of amino acids (AA) has rapidly become a powerful tool in studies of food web architecture, resource use, and biogeochemical cycling. However, applications to avian ecology have been limited because no controlled studies have examined the patterns inAAisotope fractionation in birds. We conducted a controlledCSIAfeeding experiment on an avian species, the gentoo penguin (Pygoscelis papua), to examine patterns in individualAAcarbon and nitrogen stable isotope fractionation between diet (D) and consumer (C) (Δ¹³C_C‐Dand Δ¹⁵N_C‐D, respectively). We found that essentialAAδ¹³C values and sourceAAδ¹⁵N values in feathers showed minimal trophic fractionation between diet and consumer, providing independent but complimentary archival proxies for primary producers and nitrogen sources respectively, at the base of food webs supporting penguins. Variations in nonessentialAAΔ¹³C_C‐Dvalues reflected differences in macromolecule sources used for biosynthesis (e.g., protein vs. lipids) and provided a metric to assess resource utilization. The avian‐specific nitrogen trophic discrimination factor (TDF_Glu‐Phe= 3.5 ± 0.4‰) that we calculated from the difference in trophic fractionation (Δ¹⁵N_C‐D) of glutamic acid and phenylalanine was significantly lower than the conventional literature value of 7.6‰. Trophic positions of five species of wild penguins calculated using a multi‐TDF_Glu‐Pheequation with the avian‐specificTDF_Glu‐Phevalue from our experiment provided estimates that were more ecologically realistic than estimates using a singleTDF_Glu‐Pheof 7.6‰ from the previous literature. Our results provide a quantitative, mechanistic framework for the use ofCSIAin nonlethal, archival feathers to study the movement and foraging ecology of avian consumers.

    

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4. Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues

   https://doi.org/10.1002/rcm.8408  

   Hetherington, Elizabeth D. ; Kurle, Carolyn M. ; Ohman, Mark D. ; Popp, Brian N. ( April 2019 , Rapid Communications in Mass Spectrometry)

   It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound‐specific isotope analysis of amino acids (CSIA‐AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acidδ¹⁵N values.

   We evaluated the effects of chemical preservatives on bulk tissueδ¹³C andδ¹⁵N and amino acidδ¹⁵N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species,Calanus pacificusandEucalanus californicus, which were preserved in formaldehyde for 24–25 years.

   Tissues in formaldehyde‐ethanol had higher bulkδ¹⁵N values (+1.4,D. gigas; +1.6‰,T. albacares), higherδ¹³C values forD. gigas(+0.5‰), and lowerδ¹³C values forT. albacares(−0.8‰) than frozen samples. The bulkδ¹⁵N values from copepods were not different those from frozen samples, although theδ¹³C values from both species were lower (−1.0‰ forE. californicusand −2.2‰ forC. pacificus) than those from frozen samples. The mean amino acidδ¹⁵N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanineδ¹⁵N values were altered to a larger extent (range: 0.5–4.5‰).

   The effects of preservation on bulkδ¹³C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulkδ¹⁵N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation ofδ¹⁵N values used in ecological studies. The preservation effects on amino acidδ¹⁵N values were also mostly minimal, mirroring bulkδ¹⁵N trends, which is promising for future CSIA‐AA studies of archived specimens. However, there were substantial differences in phenylalanine and valineδ¹⁵N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

    

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5. Meta‐analysis of primary producer amino acid δ 15 N values and their influence on trophic position estimation

   https://doi.org/10.1111/2041-210X.13678  

   Ramirez, Matthew D. ; Besser, Alexi C. ; Newsome, Seth D. ; McMahon, Kelton W. ( August 2021 , Methods in Ecology and Evolution)

   Compound‐specific stable isotope analysis of individual amino acids (CSIA‐AA) has emerged as a transformative approach to estimate consumer trophic positions (TP_CSIA) that are internally indexed to primary producer nitrogen isotope baselines. Central to accurate TP_CSIAestimation is an understanding of beta (β) values—the differences between trophic and source AA δ¹⁵N values in the primary producers at the base of a consumers’ food web. Growing evidence suggests higher taxonomic and tissue‐specificβvalue variability than typically appreciated.

   This meta‐analysis fulfils a pressing need to comprehensively evaluate relevant sources ofβvalue variability and its contribution to TP_CSIAuncertainty. We first synthesized all published primary producer AA δ¹⁵N data to investigate ecologically relevant sources of variability (e.g. taxonomy, tissue type, habitat type, mode of photosynthesis). We then reviewed the biogeochemical mechanisms underpinning AA δ¹⁵N andβvalue variability. Lastly, we evaluated the sensitivity of TP_CSIAestimates to uncertainty in meanβ_Glx‐Phevalues and Glx‐Phe trophic discrimination factors (TDF_Glx‐Phe).

   We show that variation inβ_Glx‐Phevalues is two times greater than previously considered, with degree of vascularization, not habitat type (terrestrial vs. aquatic), providing the greatest source of variability (vascular autotroph = −6.6 ± 3.4‰; non‐vascular autotroph = +3.3 ± 1.8‰). Within vascular plants, tissue type secondarily contributed toβ_Glx‐Phevalue variability, but we found no clear distinction among C₃, C₄and CAM plantβ_Glx‐Phevalues. Notably, we found that vascular plantβ_Glx‐Lysvalues (+2.5 ± 1.6‰) are considerably less variable thanβ_Glx‐Phevalues, making Lys a useful AA tracer of primary production sources in terrestrial systems. Our multi‐trophic level sensitivity analyses demonstrate that TP_CSIAestimates are highly sensitive to changes in bothβ_Glx‐Pheand TDF_Glx‐Phevalues but that the relative influence ofβvalues dissipates at higher trophic levels.

   Our results highlight that primary producerβvalues are integral to accurate trophic position estimation. We outline four key recommendations for identifying, constraining and accounting forβvalue variability to improve TP_CSIAestimation accuracy and precision moving forward. We must ultimately expand libraries of primary producer AA δ¹⁵N values to better understand the mechanistic drivers ofβvalue variation.

    

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