Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Summary

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Abstract

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (¹³C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall ¹³C enrichment in fatty acids derived from the ¹³C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M⁺) of each labeled FA by one or more mass units (M⁺¹, M⁺², M⁺³, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Introduction

In a cryptic habitat such as soil, trophic relationships are difficult to address and are further restricted by the small size of the fauna. The last decade has seen advances in biochemical ecology, particularly in the use of fatty acids as biomarkers for defining feeding strategies of the soil fauna under field conditions^1,2,3. This is based on the fact that fatty acids from resources can be incorporated in consumer tissue as entire molecules, a process termed dietary routing⁴. Transfer of fatty acids has been reported over three trophic levels, i....

Protocol

The described protocol does not fall under the competence of Animal Ethics. However, when people adapt the described protocols to higher animals, take care that the institutional Animal Ethics committee approved the protocol for animal handling.

1. Cultivation of Animals

NOTE: All explained experimental steps are based on well-established protocols^26,27,28. Biotests in the laboratory need a continuous supply of easily culturable organisms. Here, the Collembola species Protaphorura fimata (Gisin, 1952) and Hete....

Results

Fresh weight and lipid content of Collembola
In the course of the described experiment, the content in NLFAs and PLFAs did not change significantly over time, whereas the fresh weight of specimens increased slightly but not significantly²⁴. Both parameters indicate a good level of physical fitness of the Collembola specimens. Be aware to investigate Collembola's fresh weight and lipid content throughout the experiment corresponding to the sampl.......

Discussion

Isotopologue profiling

A detailed analysis of the quantitative aspects in ¹³C distribution in FAs needs cutting-edge technology to assign carbon partitioning in food webs. The present work employed isotopologue profiling to assess the ¹³C/¹²C ratios in common FA biomarkers for tropic interactions. This method is well established for amino acid analysis by liquid chromatography (LC-MS) and was applied for investigations of carbon metabolism in p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
neoLab-Round jars	neoLab	2-1506	69 x 40 mm, 10 pacs/pack
Charcoal activated	Carl Roth	X865.1	p.a., powder, CAS No. 7440-44-0
Alabaster Dental	RÖHRICH-GIPSE	---	http://www.roehrich-gipse.de/dentalgipse.php
Chloroform	Carl Roth	7331.1	HPLC ≥ 99,9 %
Methanol	Carl Roth	P717.1	HPLC ≥ 99,9 %
Hexan	Carl Roth	7339.1	HPLC ≥ 98 %
tert-Butyl methyl ether (MTBE)	Carl Roth	T175.1	HPLC ≥ 99,5 %
Aceton	Carl Roth	7328.2	HPLC ≥ 99,9 %
NaOH	Carl Roth	6771.1	p.a. ≥99 %, in pellets
di-Natriumhydrogenphosphat	Carl Roth	P030.1	p.a. ≥99 % , water free
Na-dihydrogenphosphat Dihydrat	Carl Roth	T879.1	p.a. ≥99 %
Hypochloric acid (6 N)	VWR International	26,115,000	AVS TITRINORM vol. solution
Bond Elut (Columns)	Agilent Tech.	14102037	HF Bond Elut-SI, 500 mg, 3 mL, 50/PK
Präparatengläser Duran	Glasgerätebau Ochs	135215	Ø 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket
Supelco 37 Component FAME Mix	Sigma-Aldrich	47885-U Supelco	10 mg/mL in methylene chloride, analytical standard
FlowMesh	Carl Roth	2796.1	Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing
Bacterial Acid Methyl Ester (BAME) Mix	Sigma-Aldrich	47080-U Supelco	10 mg/mL in methyl caproate, analytical standard
Methyl nonadecanoate	Sigma-Aldrich	74208	analytical standard ≥ 98.0 %
Hexadecanoic acid-1-13C (Palmitic)	Larodan Fine Chemicals	78-1600	GC ≥ 98.0 % (13C: 99.0 %)
RVC 2-25 CDplus	Martin Christ Gefrier-trocknungsanlagen		Compact benchtop midi concentrator
Alpha 2-4 LDplus	Martin Christ Gefrier-trocknungsanlagen		Drying manifold
MZ 2C NT	Vacuubrand GMBH		Vacuum pump
Roto-Shake Genie	Scientific Industries		Combined rocking and rotating device
XP64 Micro Comparator	Mettler Toledo		Super high precision balance
GC-System 7890A	Agilent Tech.		Gas chromatograph
7000 GC/MS Triple Quad	Agilent Tech.		Triple Quad mass spectrometer
7683B Series Injector	Agilent Tech.		Sample injector
Heraeus Multifuge 3SR+	Thermo Scientific		Centrifuge with 10 ml tube rotor