Name: fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers
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Description: Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

The financial support of R. Menzel and L. Ruess by the Deutsche Forschungsgemeinschaft (RU RU780/11-1) is gratefully acknowledged. R. Nehring was funded by RU 780/10-1. Finally, we are extremely thankful to Dr. Hazel Ruvimbo Maboreke for proofreading our manuscript.

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 protocols26,27,28. Biotests in the la...

<ol>
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F., David, F., Brunengraber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+13C+mass+isotopomer+distributions+for+natural+stable+isotope+abundance.">Correction of 13C mass isotopomer distributions for natural stable isotope abundance.</a> J Mass Spectrom. 31 (3), 255-262 (1996).</li><li>Heuner, K., Eisenreich, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+metabolism+of+legionella+by+isotopologue+profiling.">The intracellular metabolism of legionella by isotopologue profiling.</a> Methods Mol Biol. 954, 163-181 (2013).</li><li>Willenborg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+pivotal+carbon+metabolism+of+Streptococcus+suis+serotype+2+under+ex+vivo+and+chemically+defined+in+vitro+conditions+by+isotopologue+profiling.">Characterization of the pivotal carbon metabolism of Streptococcus suis serotype 2 under ex vivo and chemically defined in vitro conditions by isotopologue profiling.</a> J Biol Chem. 290 (9), 5840-5854 (2015).</li><li>Menzel, R., Ngosong, C., Ruess, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopologue+profiling+enables+insights+into+dietary+routing+and+metabolism+of+trophic+biomarker+fatty+acids.">Isotopologue profiling enables insights into dietary routing and metabolism of trophic biomarker fatty acids.</a> Chemoecology. 27 (3), 101-114 (2017).</li><li>Buse, T., Ruess, L., Filser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+trophic+biomarkers+for+Collembola+reared+on+algal+diets.">New trophic biomarkers for Collembola reared on algal diets.</a> Pedobiologia. 56 (3), 153-159 (2013).</li><li>Hutson, B. 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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...

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 conditions1,2,3. 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.e., from fungi to nematodes to Collembola5. Recently, the predatory fauna was considered6,7 and the first reviews on fatty acids as trophic markers in soil food webs have been published8,9.
More detailed information on trophic interactions is attained by fatty acid stable isotope probing (FA-SIP). The determination of 13C/12C ratios in fatty acids in diets and consumers can ascribe binary links and estimate the associated carbon flow, and has been employed in terrestrial, fresh water, and marine food webs10,11,12,13. The basic assumption is that dietary routed fatty acids are not subject to enzymatic processes; therefore, their 13C signal, i.e., the 13C/12C ratio of the fatty acid, in the consumer is similar to that in the diet1. However, a gradual depletion of the 13C signature up the food chain has been reported in aquatic systems, thereby hindering broad application of FA-SIP in trophic studies14,15,16. Moreover, knowledge in the lipid metabolism in most invertebrates in terrestrial food webs is still limited.
An understanding of the lipid metabolism pathways in consumers is essential for the usage of trophic marker fatty acids as means for the determination of the quantitative carbon flow in food web ecology. With this in mind, 13C-isotopologue profiling, which in principle can be applied for investigation of the carbon metabolism of any biological system17, is a promising method. Following the introduction of a 13C-labelled carbon substrate, the distribution of the 13C in the metabolic network is traceable since the generated metabolic products in the consumer show a specific isotopologue distribution. This can be assessed by quantitative nuclear metabolic resonance spectroscopy18,19 or mass spectrometry20,21, with the latter favored in biological samples with low biomass due to its higher sensitivity.
Although isotopologue profiling has been successfully applied to amino acids and provided insight into the in vivo carbon metabolism of bacterial pathogens17,22,23, its implementation in fatty acids has lagged behind. The first detailed analysis on the fate of a stable isotope labelled precursor fatty acid, its dietary routing or degradation via &#946;-oxidation, in soil invertebrate consumers, was recently performed by Menzel et al.24. Here, the methodological basics for incorporation experiments with 13C labelled fatty acids followed by isotopologue analysis of key descendants in frequent soil invertebrates, the Collembola, are provided. These microarthropods are a good model group as they form important components of the soil food web and are well investigated for their trophic marker fatty acids8,25.
An understanding of the lipid metabolism pathways in consumers is essential for the usage of trophic marker fatty acids as means for the determination of the quantitative carbon flow in food web ecology. The present protocol gives the design and set up for a laboratory feeding experiment, and the biochemical procedures for extraction and methylation of dominant lipids fractions (neutral lipids, phospholipids) from Collembola. It demonstrates how the isotopologue composition of fatty acids is analyzed by mass spectrometry and describes the related formula and calculations. This procedure results in: (i) the ratios of isotopologues exceeding the mass of the parent ion (i.e., the fatty acid molecular ion M+) by one or more mass units (M+1, M+2, M+3, etc.) and (ii) the overall 13C enrichment in fatty acids derived from the 13C labelled precursor. Although used for Collembola, this approach can generally be applied to any other predator-prey interaction on the premise that these are culturable in sufficient quantity under controlled conditions to ensure a successful label uptake and subsequent verification.

<table>
	<tbody>
		<tr>
			<td>neoLab-Round jars</td>
			<td>neoLab</td>
			<td>2-1506</td>
			<td>69 x 40 mm, 10 pacs/pack</td>
		</tr>
		<tr>
			<td>Charcoal activated</td>
			<td>Carl Roth</td>
			<td>X865.1</td>
			<td>p.a., powder, CAS No. 7440-44-0</td>
		</tr>
		<tr>
			<td>Alabaster Dental</td>
			<td>R&#214;HRICH-GIPSE</td>
			<td>---</td>
			<td>http://www.roehrich-gipse.de/dentalgipse.php</td>
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		<tr>
			<td>Chloroform</td>
			<td>Carl Roth</td>
			<td>7331.1</td>
			<td>HPLC &#8805; 99,9 %</td>
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		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>P717.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Hexan</td>
			<td>Carl Roth</td>
			<td>7339.1</td>
			<td>HPLC &#8805; 98 %</td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether (MTBE)</td>
			<td>Carl Roth</td>
			<td>T175.1</td>
			<td>HPLC &#8805; 99,5 %</td>
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		<tr>
			<td>Aceton</td>
			<td>Carl Roth</td>
			<td>7328.2</td>
			<td>HPLC &#8805; 99,9 %</td>
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		<tr>
			<td>NaOH</td>
			<td>Carl Roth</td>
			<td>6771.1</td>
			<td>p.a. &#8805;99 %, in pellets</td>
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		<tr>
			<td>di-Natriumhydrogenphosphat</td>
			<td>Carl Roth</td>
			<td>P030.1</td>
			<td>p.a. &#8805;99 % , water free</td>
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		<tr>
			<td>Na-dihydrogenphosphat Dihydrat</td>
			<td>Carl Roth</td>
			<td>T879.1</td>
			<td>p.a. &#8805;99 %</td>
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		<tr>
			<td>Hypochloric acid (6 N)</td>
			<td>VWR International</td>
			<td>26,115,000</td>
			<td>AVS TITRINORM vol. solution</td>
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		<tr>
			<td>Bond Elut (Columns)</td>
			<td>Agilent Tech.</td>
			<td>14102037</td>
			<td>HF Bond Elut-SI, 500 mg, 3 mL, 50/PK</td>
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		<tr>
			<td>Pr&#228;paratengl&#228;ser Duran</td>
			<td>Glasger&#228;tebau Ochs</td>
			<td>135215</td>
			<td>&#216; 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket</td>
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		<tr>
			<td>Supelco 37 Component FAME Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47885-U Supelco</td>
			<td>10&#160;mg/mL in methylene chloride, analytical standard</td>
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		<tr>
			<td>FlowMesh</td>
			<td>Carl Roth</td>
			<td>2796.1</td>
			<td>Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing</td>
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			<td>Bacterial Acid Methyl Ester (BAME) Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47080-U Supelco</td>
			<td>10&#160;mg/mL in methyl caproate, analytical standard</td>
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		<tr>
			<td>Methyl nonadecanoate</td>
			<td>Sigma-Aldrich</td>
			<td>74208</td>
			<td>analytical standard &#8805; 98.0 %</td>
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		<tr>
			<td>Hexadecanoic acid-1-13C (Palmitic)</td>
			<td>Larodan Fine Chemicals</td>
			<td>78-1600</td>
			<td>GC &#8805; 98.0 % (13C: 99.0 %)</td>
		</tr>
		<tr>
			<td>RVC 2-25 CDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Compact benchtop midi concentrator</td>
		</tr>
		<tr>
			<td>Alpha 2-4 LDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Drying manifold</td>
		</tr>
		<tr>
			<td>MZ 2C NT</td>
			<td>Vacuubrand GMBH</td>
			<td></td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Roto-Shake Genie</td>
			<td>Scientific Industries</td>
			<td></td>
			<td>Combined rocking and rotating device</td>
		</tr>
		<tr>
			<td>XP64 Micro Comparator</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Super high precision balance</td>
		</tr>
		<tr>
			<td>GC-System 7890A</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Gas chromatograph</td>
		</tr>
		<tr>
			<td>7000 GC/MS Triple Quad</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Triple Quad mass spectrometer</td>
		</tr>
		<tr>
			<td>7683B Series Injector</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Sample injector</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Centrifuge with 10 ml tube rotor</td>
		</tr>
	</tbody>
</table>

fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

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.

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&#39;s fresh weight and lipid content throughout the experiment corresponding to the...

Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

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

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.

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

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

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