Effect of Different Derivatization Protocols on the Calculation of Trophic Position Using Amino Acids Compound-Specific Stable Isotopes

Abstract

Amino acids compound-specific nitrogen stable isotope analysis (AA-CSIA) is an emerging tool in ecology for understanding trophic system dynamics. While it has been successfully used for several independent studies across a range of environments and study locations, researchers have encountered calculation issues for determining trophic position values. Most studies introduce modifications to the constants of trophic position equation calculations, but then fail to account for the equation variations when comparing across separate research studies. The broad acceptance of this approach is anchored in an underlying presumption that no addition of the exogenous nitrogen atom occurs in the different methods; and therefore, such variations should not affect the outcome. In this paper, we evaluate the use of the EZfaast amino acid derivatization kit (chloroformate) and compare it to the isotopic results of two other derivatization methods. We highlight new considerations for working with AA-CSIA that might account for some of the variations in the results and lead researchers to modify constants in the equation. This study concludes that developing unique constants per derivatization method is required to have more accurate cross-study comparisons of trophic positions.

Symbiodiniaceae Are the First Site of Heterotrophic Nitrogen Assimilation in Reef-Building Corals

“…The above observations suggest that δ 13 C and δ 15 N-AAs of coral tissue must be combined with plankton abundance and environmental data before they can be used as reliable proxies for heterotrophy. Although δ 13 C and δ 15 N-AAs values cannot be used to compare reef and aquarium-reared corals, the TP Glx-Phe should be independent of the environmental isoscape ( 28 , 30 ), and should reveal the trophic status of corals. Compared to aquarium conditions (AUT, MIX, and HET), the TP Glx-Phe value of S. pistillata reef samples was similar to the mixotrophic and heterotrophic conditions, suggesting that this species should rely on a mixotrophic diet, even though the corals were collected at shallow depths, where autotrophy should provide most of their energetic requirements.…”

“…1B ) ( 28 , 29 ). Therefore, regardless of the isoscape, the δ 15 N difference between glutamic acid and phenylalanine was used to calculate the trophic position of an organism (TP Glx-Phe ), i.e., the level it occupies in a food web ( 28 , 30 ). A TP Glx-Phe of 1 represents autotrophs, whereas higher TP Glx-Phe values represent primary and secondary consumers ( 28 ).…”

The power and pitfalls of amino acid carbon stable isotopes for tracing the use and fate of basal resources in food webs

“…Recent work indicates that the δ 13 C-EAA patterns of Symbiodiniaceae may be distinctive from other free living dinoflagellates (Stahl et al 2023). Baseline δ 13 C-EAA values often vary between the symbionts and coral tissue but whether this translates to differing δ 13 C-EAA patterns is unclear (Martinez et al 2020, 2021. Although sponge microbial symbionts have not been successfully cultivated (Pita et al 2018), sponge tissue and their assumed symbiotic microbes have been separated…”

Symbiont starvation affects the stability of the coral–Symbiodiniaceae symbiosis

“…The nitrogen and carbon isotopic compositions of amino acids were determined by gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS). The acidhydrolyzed host and symbiont samples were first derivatized using the Ezfaast kit before isotopic analysis was performed, according to Martinez et al (2020b). Briefly, 3.5 mg of the hydrolyzed samples were derivatized with the Ezfaast kit with a slight modification of replacing reagent 6 with dichloromethane as solvent.…”

“…Stable isotope ratios were expressed in standard d notation, being atmospheric N 2 (air). The trophic position (TP glu-phe ) was calculated using glutamic acid and phenylalanine with the predefined equation of Chikaraishi et al (2009) with the constants from Martinez et al (2020b).…”