Archives of Biochemistry and Biophysics 589 (2016) 27e37

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Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

Review article

The Caenorhabditis elegans lipidome

A primer for lipid analysis in Caenorhabditis elegans
Michael Witting a, *, Philippe Schmitt-Kopplin a, b
a
Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Ingolsta €dter
Landstraße 1, 85764 Neuherberg, Germany
b €t München, Wissenschaftszentrum Weihenstephan, Alte Akademie 10, 85354
Lehrstuhl für Analytische Lebensmittelchemie, Technische Universtita
Freising-Weihenstephan, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Lipids play important roles in biology, ranging from building blocks of membranes to signaling lipids. The
Received 14 April 2015 nematode and model organism Caenorhabditis elegans has been used to explore lipid metabolism and
Received in revised form several techniques for their analysis have been employed. These techniques include different possibilities
2 June 2015
ranging from visualization of lipid droplets, analysis of total fatty acids to analysis of complex lipids using
Available online 10 June 2015
lipidomics approaches. Lipidomics evolved from metabolomics, the latest off-spring of the “omics”-
technologies and aims to characterize the lipid content of a given organism or system. Although being an
Keywords:
extensively studied model organism, only a few applications of lipidomics to C. elegans have been re-
Lipidomics
Caenorhabditis elegans
ported to far, but the number is steadily increasing with more applications expected in the near future.
Lipid analysis This review gives an overview on the C. elegans lipidome, lipid classes it contains and ways to analyze
Model organism them. It serves as primer for scientists interested in studying lipids in this model organism and list
methods used so far and what information can be derived from them. Lastly, challenges and future
(methodological) research directions, together with new methods potentially useful for C. elegans lipid
research are discussed.
© 2015 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Lipidomics, the comprehensive analysis of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Caenorhabditis elegans, a versatile model organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
The C. elegans lipidome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Fatty acids and amides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Glycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Steroids and related substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Prenol lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Others classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Applied lipids analysis methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Tools for imaging of lipids and lipid deposits in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Lipid extraction and fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Mass spectrometry based methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Challenges and future research directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Dispatch the lack of C. elegans lipid standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

* Corresponding author.
E-mail address: michael.witting@helmholtz-muenchen.de (M. Witting).

http://dx.doi.org/10.1016/j.abb.2015.06.003
0003-9861/© 2015 Elsevier Inc. All rights reserved.
28 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37

MALDI imaging to understand spatial distribution of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Knowledge transfer and standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Introduction larval stages, named L1 to L4. Under unfavorable conditions, like

overcrowding or food scarcity it can enter an alternative, non-
Lipidomics, the comprehensive analysis of lipids feeding and long-lived, developmental stage, the dauer stage
(dauer, german for enduring). After conditions ameliorate dauer
Lipids play essential roles cross all kingdoms in biology and their larvae normally develop through L4 larvae to adults without
analysis in health and disease has a long tradition. In the post- compromises in adult life span.
genomic era more emphasis is put on functional analysis of genes C. elegans is used as model organism in different research fields
and genomes using tools of functional genomics: transcriptomics, (e.g. toxicology, developmental biology, neurobiology, host-
proteomics and metabolomics, all of them studying a different pathogen interactions, aging research and others). A large array of
subpart of a cells or organisms interior. Because lipids play such an tools was developed over the past years involving automated
important role as building blocks of membranes, energy storage or worm-sorting, micro arrays for transcriptomics, imaging technol-
second messengers and signal transducer, the comprehensive ogies, transgenics or tissue specific gene inactivation [16]. Further
analysis of lipids evolved from metabolomics as individual disci- details on the biology of the worm can be found in the review by
pline called “lipidomics”. Hulme and Whitesides [17].
In contrast to general assumption that lipids represent a rather Although extensively studied, metabolomics and lipidomics
homogenous class, several tens of thousands different lipid struc- investigations on C. elegans are scarce, but number of publications
tures are possible [1]. Lipids are present in a large diversity, from related to this topic is increasing steadily. Within in this review we
ubiquitous bulk phospholipids in the membranes to highly specific aim to give a general overview on lipids present C. elegans. This
signaling lipids and span several orders of magnitude in concen- information was inferred from genetic information on lipid meta-
tration. Additionally, different lipid classes have large differences in bolism readily available since many years and results from different
polarity (e.g. PC(18:0/16:0) has a log P of 11.94 and TG(16:0/16:0/ publication. From this we deduced lipid classes and species that
16:0) a log P of 22.08) or molecular mass (e.g. palmitic acid: might be present in the worm. Furthermore, we introduce methods
255.231857, [M
H]-compared to CL(10 -[18:2(9Z,12Z)/ for their analysis and how they have been used so for analysis of the
0
18:2(9Z,12Z)],3 -[18:2(9Z,12Z)/18:2(9Z,12Z)]): 1447.964950, C. elegans lipid content. Finally, challenges and needs for successful,
[M
H]-), which dramatically complicates their analysis. future lipidomic investigations are discussed.
Today, lipidomics is mainly based on Mass Spectrometry (MS), This review serves as primer for scientists interested in
but also Nuclear Magnetic Resonance (NMR) has been employed analyzing lipids in the model organism C. elegans.
[2]. Two different kinds of MS analysis can be differentiated: (i)
shotgun lipidomics directly infuses raw lipid extracts into the MS
The C. elegans lipidome
and uses combinations of neutral loss and product ion scans to
differentiate and identify lipids and is mostly carried out on ion trap
C. elegans is able to produce a wealth of different lipids. In 2013
MS offering MSn capabilities, but also Quadropole-Time of Fligth
Zhang et al. collected “all” lipid metabolic genes present in the
(Q-ToF) instruments are employed. Major advantage of this tech-
worm by comparative genomics. In total they found 471 lipid genes
nology is its high-throughput possibility. However, missing sepa-
curated from KEGG, literature research and orthologues of human
ration of isomeric lipids is the major disadvantage of this approach.
lipid genes. 237 of 471 of these genes are conserved in humans,
(ii) Lipid profiling using Liquid ChromatographyeMass Spectrom-
mice, rats or Drosophila. More specifically 71 of these conserved
etry (LCeMS) is the second most used technique so far. The use of
genes are related to human metabolic diseases. Using the OMIM
chromatographic separation enables resolving of isomers including
database most genes were annotated by the keywords “Obesity,
cis/trans isomers [3]. In most cases, reversed phase (RP) separations
Diabetes, Metabolic disease and others”. Furthermore, 327 genes in
using C8 or C18 columns with an Acetonitrile-iso-Propanol (ACN-
C. elegans are orthologues to human disease genes, which implies
iPrOH) gradient are employed [3e5], but also application of HILIC
that lipid genes are also involved in diseases different from meta-
and normal phase separation is common [6,7]. Lipidomics is
bolic syndrome. This collection was complemented by a functional
applied in any field of biological research ranging from cell cultures
study using RNA interference (RNAi), which revealed several phe-
[8], microorganisms [9e11], plants [12,13] and mammals [14].
notypes, including growth and developmental defects also for
genes not reported so far [18]. One particular example how such
Caenorhabditis elegans, a versatile model organism genes relate to human disease genes was published by Hashmi
et al., who used the worm’s intestine as model system to study the
The soil-dwelling nematode C. elegans has become a major role of Krüppel-like factors in fat regulation, cell death and
model organism in biology. It offers several experimental advan- phagocytosis [19]. Additional the role of these factors in human
tages, including a fast reproductive cycle, a translucent body, metabolic regulation was reviewed and KLF-3 for example acts
known cell lineage and a sequenced genome, which was the first of selectively on insulin components [20].
a multicellular organism [15]. Furthermore, its hermaphroditic Although of impressive nature, this work and other genetic
nature allows raising a large number of isogenic animals in short studies gives no information on the actual chemical diversity of the
time. lipid content, which is very strongly condition dependent (e.g.
The full developmental cycle of C. elegans from eggs to fertile availability of building blocks like fatty acids). In this part we pre-
adults takes about 3 days at 20  C. The worm develops through four sent different lipid classes found in C. elegans, their biosynthetic
 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37 29

pathways and selected biological actions. Table 1 summarizes
classes present in the worm along with their commonly used ab-
breviations and LipidMaps identifiers, while Fig. 1 shows their basic
structure examples of them.
Fatty acids and amides
Except for steroids, fatty acids are one of the major building
blocks of complex lipids. C. elegans is able to synthesize a broad
variety of fatty acids on its own, including saturated (FA), mono-
and polyunsaturated (MUFA and PUFA) and mono methyl branched
fatty acids (mmBCFA). Straight chain fatty acids are synthesized
from acetyl-CoA as primer by condensation with malonyl-CoA in
contrast to mmBCFAs, which are produced from branched chain
primers derived from catabolism of branched chain amino acids.
Fatty acids biosynthesis has been extensively studied and several
elongases and desaturases and their regulation have been
described [21,22]. A 13C isotope labeling approach demonstrated
which fatty acids are directly taken up from the worm’s diet and
which are synthesized de novo. The two cyclo propane fatty acid
C17D and C19D are exclusively taken up from bacterial diet and
C16:1n7 and C18:1n7 are synthesized de novo in minor amounts of
about 5%, whereas C16:0, C18:0, C18:1n9, C18:2n6 are produced in
increasing amounts ranging from 7.2% to 19% and mmBCFAs are
exclusively synthesized de novo [23,24].
Fatty acids not only serve as building blocks for higher lipids, but
have rather important biological roles. For example g-linolenic and
stearidonic acid are required for basal immunity during infection
with Pseudomonas aeruginosa [25], while oleic acid and NHR-80/
HNF4 are needed for longevity in germline ablated worms [26].
The mmBCFA C17:0iso is essential for postembryonic growth of L1
larvae [27]. Additionally, C17:0iso and ACS-1 are shown to be
needed for inositol triphosphate (IP3) signaling for early embryonal
development [28].
Furthermore, it has been demonstrated that PUFAs are impor-
tant for correct neurotransmission in C. elegans. Watts et al.
encountered non-normal behavior of fat-3 mutants during their
studies of fatty acid metabolism [29]. Depletion of PUFAs in fat-3
mutants leads to movement deficiencies, inability to respond to
head-touch and egg laying defects similar to egl-1 mutants. Defects
in motility were rescued by arachidonic acid (C20:4n-6) and
docosahexaeonic acid (C22:6n-3), similarly expression of fat-3
under control of the neuronal promotor unc-119 could also rescued
the egl phenotype. Further experiments lead to the conclusion that
mutation of fat-3 leads to functional but not developmental defects
in the nervous system and insufficient neurotransmitter release
[30].
Composition of fatty acids also correlates with longevity. Fatty
acid profiles from different long lived mutants were measured and
by correlating relative longevity with different parameters (e.g.
chain length or degree of unsaturation) different trends became
apparent. First, the amount of MUFAs increases, while PUFAs
decrease with longevity. In parallel a trend towards shorter chain
length is observed. Results were also in accordance with suscepti-
bility to hydrogen peroxide [31].
One lipid class directly derived from fatty acids are fatty amides,
represented in C. elegans by N-acylethanolamides (NEA). This lipid
class includes the mammalian endocannabinoid anandamide and is

Table 1
Lipid classes present in C. elegans inferred from genetic information.

Lipid class Subclass Abrrev. LM ID

Sphingolipids Sphingosine SPH SP0101

Sphiganine (Dihydrosphingosine) SP0102
Sphingosine 1-phosphate S1P SP0105
Sphinganine 1-phospate SP0105
N-Acylsphingosine (Ceramide) Cer SP0201
N-Acylsphinganine (Dihydroceramide) SP0202
Ceramide 1-phosphate CerP SP0205
Sphingomyelin SM SP0301
Glucosylceramide GlcCer SP0501
Lactoyslceramide LacCer SP0501

Glycerolipids* 1-Acyl-sn-glycerol MG GL01

1,2-Diacyl-sn-glycerol DG GL02
Triacylglycerol DG GL03

Glycerophospholipids* Phosphatidic acid PA GP10

Phosphatidylcholine PC GP01
Phosphatidylserine PS GP03
Phosphatidylethanolamine PE GP02
Phosphatidylmonomethylethanolamine MMPE e
Phosphatidyldimethylethanolamine NMPE e
Phosphatidylinositol PI GP06
Cardiolipin CL GP12

Steroids and related substances Steroids ST ST0101

Dafachronic acids DA ST0403
Cholesterylester CE ST0102

Glycolipids Maradolipids Mar e

Fatty acids Straight chain fatty acids FA FA0101

Branched chain fatty acids FA FA0102
Unsaturated fatty acids FA FA0103

Fatty amides Fatty amides FA-EA FA0804

Prenol lipids Isoprenoids PR PR01
Quinonnes PR PR02

Others Ascarosides Ascr e

* Depicted class also includes fatty acids bound as alkyl or alkenyl.
30 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37

Fig. 1. Structures of lipid classes present in C. elegans (A) sphingolipids usually contain a C17iso sphingoid base. Different derivatives are possible, including S1P, Cer, CerP, SM, GlcCer
and LacCer, (B) Glycerolipids are the major energy storage in C. elegans. MG and DG also serve as precursor for other lipids. (C) Glycerophospholipids are the major lipid class found
as building blocks of biological membranes in high amounts. C. elegans is capable of synthesizing a wide range with different headgroups, e.g. PA, PC, PS, PE, MMPE, DMPE and PI. (D)
The bile acid like dafachronic acids (D7-DA shown as example) are important signaling molecules in development and nematodal longevity. (E) Maradolipids are special lipids found
in dauer larvae, only recently discovered (F) Fatty acids are used as building blocks of complex lipids, but also serve as signaling molecules, while fatty amides are important
signaling molecules, e.g. linking food to lifespan.

synthesized by acylation of the ethanolamine nitrogen of PEs fol-
lowed by cleavage of the phosphate ester bond yielding a NEA and
phosphatidic acid. Eicosapentanoyl ethanolamide is reduced upon
starvation or dietary restriction and mediates diet effects to life-
span [32]. In a metabolomics approach oleolyethanolamide was
identified to be increased in long lived worms over expressing lipl-
4, and binds to the proteins LBP-8 and NHR-80, activates target
genes of NHR-80 and NHR-49 and promotes longevity [33].
Glycerophospholipids
Glycerophospholipids, as building blocks of membranes,
represent the largest portion of the lipidome. Chemically they are
built from two fatty acids esterified to the sn1 and sn2 positions of
glycerol and different head groups attached to sn3 position. Addi-
tionally, side chains at the sn1 position can be bound by either an
acyl, alkyl or alkenyl bond. Generally fatty acids bound on the sn1
position have a low degree of unsaturation, while different fatty
acids can be found on the sn2 position. From genetic analysis
C. elegans is able to synthesize and use a high diversity of different
head groups, resulting in many different lipid classes: phosphatidic
acids (PA), phosphatidyl cholines (PC), phosphatidyl ethanolamines
(PE), phosphatidyl serines (PS), phosphatidyl glycerols and glycerol
phosphates (PG and PGP), and phosphatidyl inositols (PI).
Furthermore, cardiolipins (CL) are found in mitochondrial mem-
branes and N-mono- and di-methyl phosphatidyl ethanolamines
(MMPE and DMPE) are intermediates in the synthesis of PCs from
PEs present in certain mutants or knock-downs [34]. For the three
major glycerophospholipid classes (PC, PE and PS) biosynthesis
pathways are strongly connected (Fig. 2B). PCs can be synthesized
on different routes. First, the Kennedy pathway (blue in Fig. 2B)
uses dietary choline or phosphoryl choline derived from the
phosphobase methylation pathway [34,35] (red in Fig. 2B) and its
activated form CDP choline for coupling to diacylglycerols (DG).
There is some evidence for the existence of the BremereGreenberg
pathway in C. elegans [36]. PS synthesized from PC by exchange of
the choline head group with serine catalyzed by PSSY-1 [37] and PE
is derived from PS by decarboxylation. As major building blocks of
membranes, glycerophospholipid strongly influence mechanical
properties of cells and membrane fluidity. In the previous
 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37 31

Fig. 2. Examples of different lipid synthesis pathway present in C. elegans. Genes encoding lipid enzymes are depicted in red next to the reaction the products catalyze. (A) A vast
amount of different fatty acids with different chain length and degree of saturation can be produced by C. elegans on its own, although it utilizes fatty acids derived from bacterial
diet directly. Exceptions are mmBCFA, which are almost exclusively synthesized by the worm itself. (B) Glycerophospholipids are the major building blocks of biological membranes
and the synthesis of different classes is strongly interwoven. The shaded orange region shows the phosphobase methylation pathway producing phosphoryl choline from serine. The
Kennedy pathway (blue region) directly utilizes dietary choline for synthesis of PC. Lastly, PS is produced from PC by exchanging the headgroups. Some evidence for existence of the
BremereGreenberg pathway (green region) has been found in C. elegans.

paragraph PUFAs were linked to correct neurotransmission. Newer
work shows that PUFAs bound to specific glycerophospholipids are
needed for neuronal cell mechanics and touch sensation. fat-3, fat-
4, fat-1 and elo-1 had reduced touch response compared to wild
type worms. Mutants were supplemented with either arachidonic
acid (C20:4n-6), eicosopentaenoic acid (C20:5n-3) or both. Inter-
estingly, only when both were supplied mutants behaved like the
wild type and in the next step it was evaluated if PUFAs bound to
phospholipids or in another form are needed for phenotypic rescue.
The enzymes MBOA-7 and MBOA-6 are responsible for integrating
PUFAs into PIs or PCs, PEs and PSs. mboa-7 mutants and mboa-
6(RNAi) (knockout of mboa-6 is lethal) were fed either with
PE(18:0/20:4), PC(18:0/20:4) or PS(18:0/20:4) or different combi-
nations. Only PE(18:0/20:4) and PS(18:0/20:4) showed significant
enhancement. Lastly, isolated touch receptor neurons (TRNs) of
wild type and fat-1; fat-4 animals showed different membrane
mechanics [38].
Sphingolipids
Sphingolipids play important roles as both structural units and
signaling molecules. Structurally, they are built from a sphingoid
32 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37
base, derived from condensation of a fatty acid (usually C16:0) with
serine and N-linked fatty acid. C. elegans on the contrary uses C15:0
and produces a C17:0iso branched chain sphinganine base
(d17:0iso-SPA). However under certain conditions, like RNAi of the
gene let-767, glucosylceramides also contain C16 and hydroxy-
C17:0 iso sphingoid bases [39]. Interestingly, N-linked fatty acid
side chains contain 2-hydroxy fatty acids, mostly C22:0-OH [40].
The biosynthesis pathway with all related genes is shown in Fig. 2C.
Previous work showed that elo-5 loss-of-function mutations
lead to arrest in the early L1 stage. Zhu et al. demonstrated that this
mutation leads to disruption of a novel sphingolipid-TORC1
pathway needed for normal development. Several genes for syn-
thesis of glucosyl ceramides (GlcCer) from d17:iso-SPA were tested
and exogenous supplementation with specific sphingolipids could
rescued the phenotype of elo-5(
) and splt-1(RNAi), but not of fath-
1(RNAi) and cgt-1(
), cgt-3(
) (fath-1 encodes for fatty acid 2-
hydroxylase and cgt-1 and cgt-3 for ceramide glucosyl trans-
ferases), which suggests that d17iso-GlcCer is the mediator in this
pathway. Using a genetic screen a loss-of-function mutation of nprl-
3 was detected to abolish this effect [41]. These results directly link
mmBCFA to a new signaling pathway. Other publications demon-
strated that ceramides are required for anoxia protection [42],
autophagy dependent life span extension [43] or radiation induced
apoptosis [44].
Glycerolipids
Excessive energy in C. elegans is stored in form of lipid droplets,
containing tri acyl glycerols (TGs), which are primarily located in
epidermal and intestinal cells (up to 35% of body dry mass) and to a
lesser extends in form of glycogen (3.3% of body dry mass). Lipid
storage vesicles are not overlapping with lysosome-related organ-
elles [45]. For mobilization of energy from these resources the
worm harbors several lipases, some also linked to longevity. lipl-4
for example is needed for induction of autophagy and is also
required for life span extension in germ line ablated animals [46].
Similarly, also diacylglycerol lipases regulate lifespan [47]. Fat
storages are mobilized during starvation, for example dauer larvae
accumulate fat storages, which are utilized during prolonged star-
vation [48,49]. The full complement of peroxisomal and b-oxida-
tion genes is present in C. elegans for generation of energy from fat
storages, including a functional methylmalonyl-CoA epimerase for
degradation of mmBCFAs [50,51].
Steroids and related substances
The worm is a sterol auxotroph and therefore sterols have to be
supplied exogenously by the diet, whereby different sterols can
serve the needs of C. elegans. Interestingly, the needed amount of
sterols is too low to have structural roles, but is rather needed for
synthesis of steroid signaling molecules [52]. Indeed, development
is regulated by bile acid like steroid hormones called dafachronic
acids [53]. Several different forms of dafachronic acid have been
identified [53,54], but the exact biosynthesis pathway is still not
completely known. Dafachronic acids not only regulate develop-
ment, but are also required for signaling in longevity. For example
the long life of germline ablated animals involves the presence of
the DAF-9/DA/DAF-12 pathway [55e57]. Furthermore, dietary re-
striction mediated longevity also relies on dafachronic acid
signaling, but instead of DAF-12 its close homolog NHR-8 is
required and let-363/mTOR is essential for this mediation [58].
Beside this bile acid like steroid hormones C. elegans contains
different other sterols, where by the actual content of individual
compounds is dependent on the sterol supplementation [59,60].
Glycolipids
A special class of glycolipids was recently discovered in dauer
larvae of C. elegans: 6,60 -diacyltrehaloses, called maradolipids.
Interestingly most of the detected lipids from this class contained at
least one mmBCFA side chain. Further experiments showed that
these special lipids are part of the dauer body structural unit, the
microvilli in the lumen. Using elo-5 RNAi in a daf-2 background or
the daf-2;DDtps mutant microvilli size is reduced or completely
missing [61].
Prenol lipids
Prenol lipids are derived from Acetyl-CoA utilized in the
mevalonate pathway. In contrast to other animals, C. elegans lacks
the cholesterol producing branch of the mevalonate pathway, but
all others are highly conserved. This makes the worm an ideal
model organism for the study of the non-cholesterol producing
branches. Knockdown of any enzyme of the main branch producing
Franesyl-PP from Acetyl-CoA leads to embryonic lethality. Likewise,
inhibition of HMG-CoA reductase by statins also leads to embryonic
lethality [62]. Important functions of prenol lipids are derivatiza-
tion of adenosine in tRNAs for stabilization of codoneanticodon
interactions [63], production of coenzyme Q (although also sup-
plied by bacterial diet) [64] or protein modification [65]. For further
information on the mevalonate pathway in C. elegans refer to the
review by Rauthan and Pilon [66].
Others classes
Ascarosides are a special class of lipid derived molecules, which
have different functions, from dauer inducing pheromone (dau-
mone) [67], male attractants [68] to signaling of aggregation [69].
Ascaroside signaling integrates building blocks from fatty acid,
amino acid metabolic and other pathways to form a modular library
of pheromones [70,71]. Peroxisomal b-oxidation is important for
synthesis of ascarosides and requires daf-22 and dhs-28 for pro-
duction of short chain fatty acids from very long chain fatty acid
precursors. More recently mutation in the acyl-CoA oxidases acox-
1, -2 and -3 has been shown to lead to defects in ascaroside
signaling [72]. More information on this special class can be found
in the recent review of Frank Schro €der [71].
Applied lipids analysis methods
C. elegans produces a vast amount of lipids covering a large
combinatorial and chemical space and the question rises how this
diversity can be accessed analytically? In the same way as lipids
also methods for their analysis are very diverse, ranging from im-
aging methods, analysis of single classes (e.g. fatty acid analysis
with GC) or profiling of the (complete) lipid content using MS based
technologies. Plenty of work has been carried out on the level of
fatty acids. However, as examples above have shown not only
composition of fatty acids but also their origin or relation to com-
plex lipids is important. Comprehensive analysis of intact lipids
only recently joined the C. elegans toolbox.
Still, several analytical chemistry methods have been used, on
which the current technology is built on. Major analysis technolo-
gies are imaging of lipid deposits in C. elegans using fluorescent
dyes or other technologies, analysis of lipid fractions with TLC and
analysis of fatty acid composition with GC and GCeMS. However,
the two new MS based technologies, shotgun lipidomics and
LCeMS based lipid profiling, are catching up. Depending on the
nature of the biological question that needs to be answered
different approaches or technologies might be selected. The
 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37
following paragraphs gives an overview on methods employed
with C. elegans so far and compares their advantages and disad-
vantages. Fig. 3 shows a flow chart with different possibilities for
analysis of lipids and Table 2 compares their advantages and
disadvantages.
Tools for imaging of lipids and lipid deposits in C. elegans
Classically, lipid deposits are labeled with fluorescent dyes that
concentrate in hydrophobic environments. The phenoxazone dye
Nile red or BODIPY labeled fatty acids are used for imaging of lipid
deposits in cell lines or C. elegans, by mixing with the worm’s diet
[73,74]. However, broader analysis indicated that these dyes do not
label major fat storages in C. elegans, instead fixation and staining
with oil red O correlated well with biochemical analysis of lipid
content [45]. Imaging in live animals is a useful tool to follow in-
dividuals over time. Choline containing metabolites and lipid were
imaged in live C. elegans using Stimulated Raman Scattering (SRS)
together with isotope-based metabolic labeling, that shifted the
NeH (NeD) bond vibration to 2100 cm
1 in the cell silent Raman
window [75]. Similarly, alkyne-tagged small biomolecules have
been used in conjunction with SRS for live-cell imaging, including
C. elegans. 5-Ethynyl-20 -deoxyuridine, 5-Ethynyl uridine, L-Homo-
propargylglycine, Propargylcholine and 17-Octadecynoic acid were
used to follow de novo synthesis of DNA, RNA, proteins, phospho-
lipids or triacylglycerols respectively [76]. Another example made
use of SRS together with phenyl-diyne cholesterol, which is stable
and permits specific detection, to investigate cholesterol storage in
wild type worms and chup-1 mutants, which are deficient in
cholesterol uptake [77]. Though for these methods labels are
needed, whereas coherent anti-Stokes Raman scattering (CARS) is
not dependent on prior labeling. CARS was compared with different
fat labeling methods under different conditions, which revealed
that CARS is the method of choice, if available [78]. Several other
studies reported to use of CARS as label-free approach to follow
Fig. 3. Different possibilities for analysis of lipids and lipid deposits in C. elegans have been described. This flow chart shows all major techniques and approaches with example
literature. Different combinations of steps can be employed to reach an optimal result, e.g. extraction, fraction and measurement of a specific class or fraction.
33
lipid metabolism in C. elegans [79e81].
However, all these methods give only very unspecific informa-
tion on total lipid classes, but not on individual lipid species. MS
based imaging is interesting alternative for metabolite and lipid
imaging directly in tissues without the necessity of prior labeling.
Matrix Assisted Laser Desorption IonizationeMass Spectrometry
(MALDIeMS) and Time of Flight-Secondary Ion Mass Spectrometry
(ToF-SIMS) based techniques have been used with C. elegans in two
proof-of-principle publications [82,83]. For routine application of
this technology several developments have to be made, especially
spatial resolution in MALDIeMS has to be in an acceptable range to
visualize different tissues (see also Challenges and Future research
directions).
Lipid extraction and fractionation
If individual lipids or lipid classes have to be analyzed they need
to be extracted from a C. elegans sample. The two most employed
lipid extraction methods are the Folch [84] and Bligh and Dyer
protocols [85]. Both extraction protocols are based on chloroform/
methanol, yet often replaced with protocols using MTBE [86,87],
due to the carcinogenic nature of chloroform and environmental
concerns. Comparison of extraction protocols, in which also
C. elegans eggs were included as one particular sample, exhibited
that the new method has similar yields. In contrast to chloroform,
MTBE based extractions build a two phase system with the organic
solvent on the top and cell debris on the bottom particularly useful
for robotic automation [86].
Lipid composition of C. elegans can be assessed on class level in
the simplest way by the use of thin layer chromatography (TLC).
This method is easy to use and no specialized equipment is needed
and can be performed analytically or preparative for isolation of
specific lipid classes. Quantification is achieved by general or spe-
cific staining (e.g. glycolipids [61] or phospholipids [88]) and
densitometry. Use of 2D-TLC, adding a second separation
34 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37

Table 2
Summary of advantages, disadvantages and information content of different technologies for analysis of C. elegans lipids.

Method Advantages Disadvantages Information content

Imaging (e.g.  Direct analysis  No information on lipid composition  Localization and amount of labeled and/or detectable
Fluorescence labeling,  Non-invasive, live imaging  For analysis of specific lipids or tracers with lipids
SRS CARS) possible (CARS, SRS) tags are needed

TLC  No special equipment needed  Only limited information  Amount of lipid classes
 Fast, easy and cheap

GCeFID/GCeMS  High resolution separation of  Only amenable to volatile lipids or lipids that  Fatty acid composition
isomers can be made volatile by derivatization  Amount of isomeric fatty acids
 Absolute quantification of
different fatty acids

Shotgun lipidomics  Fast, high-throughput capable  No separation of isomers  (absolute) quantities of different lipids
(analysis time of several minutes)
 Absolute quantification possible

LCeMS  Separation of isomeric molecules  Long analysis time, typically between 15 to  Absolute or relative quantities of different lipids, also
 Absolute quantification possible 25 min for (chromatographically) separable isomers

DIeHReMS  Ultrahigh resolution allows  No differentiation between isomeric  Relative quantification of isobaric lipids
separation of isobaric molecules molecules  Exact mass and molecular formula for unknown
 Fast and high throughput capable lipids

dimension, increases resolution of this technique [61]. Solid phase
extraction (SPE) represents an alternative for lipid fractionation and
is mostly based on NH2 phases for separation of different lipid
classes [88]. Also more advance materials for isolation of specific
lipid classes (e.g. S1P) are available [89].
Information on lipid composition from these methods is limited,
but they are useful for enrichment of specific lipid classes and can
be combined with more sensitive and specialized methods, like
GCeMS or shotgun lipidomics.
Mass spectrometry based methods
Mass spectrometry is a powerful tool to decipher between
different lipid species and allows precise relative and/or absolute
quantification. It can be either used without (shotgun) or with
chromatographic separation. Based on the nature of chromatog-
raphy different lipids can be analyzed.
GC and GCeMS is the most employed techniques for fatty acid
analysis in C. elegans. Fatty acids are transferred to volatile methyl
esters by esterification of free or trans-esterification of bound fatty
acids. MS can be used in conjunction with isotopic labeling to
determine rate of de novo synthesis [23]. GCeMS is often employed
in conjunction with a prior fractionation using either TLC or SPE to
assess the fatty acid composition of specific fractions. Despite, in-
formation on specific fatty acid combinations in complex lipids is
lost. Results are usually expressed as percentages of detected fatty
acids. GC allows high resolution separations, including baseline
separation of iso and ante-iso fatty acids or different double bond
isomers. A major problem when analyzing fatty acids with GCeMS
is in source fragmentation of PUFAs under EI conditions. Using
chemical ionization can overcome this and also allows locating
double bonds in PUFAs [90,91]. A novel LC-MS based method might
replace this approach in future, offering higher sensitivity using a
charge reversal derivatization of free fatty acids with N-(4-
aminomethylphenyl)pyridinium, although not applied to
C. elegans yet [92].
The analysis of the fatty acid composition of intact lipids is
getting more important, because there is only weak correlation
between bound and free fatty acids. Liquid based techniques offer
the possibility to introduce intact lipids into the MS. Two different
approaches can be used. First, shotgun lipidomics directly infuses
the raw lipid extract without prior chromatographic separation and
analysis is performed on tandem mass spectrometers utilizing
multiple neutral losses and product ion scans to determine lipid
composition of different signals. Up to know this is the mostly
employed analysis method for C. elegans lipidomes.
Shotgun lipidomics in conjunction with multiple precursor and
neutral loss scans and data dependent acquisition (DDA) was used
for the analysis of the C. elegans lipidome. It was demonstrated that
using this technique accurate identification and quantification can
be performed in one single direct infusion experiment. The
experiment was carried out on a Q-ToF experiment equipped with
an automated nanospray chip ESI source (NanoMate) allowing long
spraying times with a minimal consumption of sample extract.
Phospholipids were extracted according to Bligh and Dyer and TAGs
were isolated by 2D-TLC as described by Matyash et al. [93]. Limits
of quantification (LOQ) in the lower nM range were achieved. In
total 90 glycerophospholipids and 35 TGs with unique molecular
formulae were detected in a single experiment. MS/MS data indi-
cated that behind each individual molecular composition of one TG
mass, 5 to 15 isobaric species can be found. Using systems of linear
equations relative abundances of each specific isobar could be
calculated [94]. The same group used multivariate analysis of high-
resolution survey MS scans for lipidomics screening on an Orbitrap
instrument similarly equipped with a NanoMate for long infusion
times. For proof of concept RNAi of two genes, encoding putative
methyl transferases, was conducted. Analysis showed presence of
MMPE and DMPE in the corresponding extracts, proofing methyl
transferase activity of the two genes called pmt-1 and pmt-2 [34].
Dedicated software tools in combination with shotgun lip-
idomics enables screening of novel lipids. The software Lip-
idXplorer with its Molecular Fragmentation Query Language
(MFQL) was used to specifically search for maradolipids in extracts
from C. elegans dauer larvae in comparison to L3 larvae. Analysis
revealed a novel class of lipids called lyso-maradolipids having only
one fatty acid moiety specifically enriched in dauer larvae.
Furthermore relative abundances of fatty acid side chains could be
calculated for marado and lyso-maradolipids [95]. Due to missing
chromatography direct infusion experiments benefit from
employing high-resolution instrumentation. Ishida et al. performed
direct infusion experiments using the NanoMate chip ESI source
coupled to a Bruker APEX III ICR-FT/MS for analysis of C. elegans PEs.
High-resolution of the employed MS differentiated between diacyl-
and alkyl-acyl PEs solely on mass [96]. DI-ICR-FT/MS hold great
promises for high throughput deep metabotyping [97] and will be
potentially applied in future C. elegans lipidomics investigations.
 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37
Several other studies likewise employed shotgun lipidomics
[28,41e43].
The major drawback of shotgun lipidomics is the missing dif-
ferentiation between isomeric lipids, for which reason chromato-
graphic separation is needed. Traditionally normal-phase
separations using silica columns and non-polar solvents for elution
(e.g. iPrOH, hexane, chloroform, ethyl acetate or mixtures) have
been used for analysis of lipid classes and was also employed to
C. elegans [28]. However it requires dedicated equipment free of
water due to immiscibility of different organic solvents with water,
which would lead bad chromatographic performance, unstable
baselines and other problems. Hydrophilic Interaction Liquid
Chromatography (HILIC) is an interesting alternative approach us-
ing water miscible organic solvents for lipid class separation [6,7].
The predominant method in lipidomics nowadays is based on
RP separation and an ACN-iPrOH gradient, which allows detection
of a broad range of different lipid classes and species within a single
run [4]. Castro et al. for example studied the impact of daf-2 mu-
tation on the metabolome and lipidome. For lipid analysis GCeMS
for total fatty acids and LC-MS for intact lipids was utilized. LC-MS
analysis was performed on a C8 column with an ACN-iPrOH
gradient. Correlation analysis between total fatty acids and
LCeMS data revealed a central role for C20:2 in lipid remodeling
between triacylglycerol’s and phospholipids [98]. We have recently
described a UPLCeMS based method for comprehensive analysis of
the C. elegans lipidome using a sub-2 mm core shell particle C18
column. This method shows very high reproducibility as evaluated
with lipid standard materials and C. elegans lipid extract, even be-
tween different column batches and days. Fig. 4 shows a typical
chromatogram derived from this method. Use of DDA allows
collection of MS/MS spectra of different lipids on a large scale
already during profiling runs [5]. Furthermore, an automated
approach for analysis of this large scale MS/MS data collection was
developed. This novel approach, called LipidFrag, is based on the in
silico fragmentation engine MetFrag. A classifier model based on
true positive and false positive annotations of neutral precursor
Fig. 4. Example chromatogram derived from a C. elegans lipid extract analyzed with the method from Witting et al. in positive ionization mode [5]. Three different clusters of lipid
features can be detected. Detected classed referred in figure are independent of ionization mode.
35
masses from MS/MS spectra of lipid standards was used to evaluate
performance and calculation of reliability of in silico fragmentation
results. Using this novel workflow ambiguous result can be filtered
out [Witting et al., unpublished].
Challenges and future research directions
Although only a limited number of investigations on compre-
hensive lipid analysis in C. elegans are published so far, much has
been achieved. The worm as model system allows easy setup of
experiments related to fat and lipid metabolism and storage and a
lot of knowledge has been collected over the last years. Still, several
lipid classes lack comprehensive measurements at the moment.
Sphingosine kinases and lyases play important roles as shown by
different studies [99], but comprehensive and reliable detection of
their educts and products is still not achieved. Menuz et al., were
able to detect C17iso sphingosine-1-phosphate and also found a 10-
fold increase in sphk-1 mutants [42]. Potentially novel sample
preparation methods can overcome this limitation, which also
enabled detection new forms of S1P in Drosophila [89]. Lastly, direct
detection and identification of intact lipids using either shotgun or
LCeMS based lipidomics will replace long isolation, derivatization
and measurement of fatty acids and will lead to new insights.
However, several points have to be raised and should be addressed
for successful future investigations.
Dispatch the lack of C. elegans lipid standards
The Metabolomics Standard Initiative (MSI) defined several
levels of confidence for identification of metabolites and lipids,
whereby the highest level (level 1) can be only achieved by com-
parison with an authentic standard under the same analytical
conditions [100]. Such standards are not commercially available for
certain lipids present in C. elegans. Especially for the maradolipids
and several dafachronic acids no commercial standards are avail-
able. Though, synthesis protocols for both have been described
36 M. Witting, P. Schmitt-Kopplin / Archives of Biochemistry and Biophysics 589 (2016) 27e37
[54,101e103]. Interestingly, ceramides with 2-OH-fatty acid are
available, but not with C17:0iso base. However, such standards
would be needed for definite identification and comparison with
ceramides present in C. elegans. Synthesis of chemical reference
standards for each individual lipid is unrealistic and not feasible,
but representative candidates from each lipid class are needed at
minimum. Normally, lipids from the same class exhibit similar
fragmentation spectra. Therefore one or several standards can
serve as proxy for identification.
Far more important is the need for suitable internal standards
for normalization and quantification. Because C. elegans is capable
of producing and using odd chain fatty acids, lipids normally
applied as internals standards like PC(17:0/17:0) are not suitable.
Isotopes labeled analogs are a possible solution (e.g. lipids with
deuterated side chains or 13C labeled side chains) to this issue, but
are either not available or very expensive.
MALDI imaging to understand spatial distribution of lipids
The nematode represents a multicellular organism with differ-
entiated tissues. These tissues vary in their lipid content and
composition. The intestine is the major storage of lipid droplets
containing TGs. Similarly composition of phospholipids may vary
between tissues. Several imaging tools have been described to
locate and analyze lipid deposits, e.g. based on fluorescent dyes or
CARS. These tools give no information on lipid composition, for
example different fatty acid side chains or their combination. So far
only phosphatidylcholine has been imaged using isotope labeling
in combination with SRS [75].
MALDIeMS based imaging is gaining popularity as alternative
tool to access lipid composition and spatial information in a single
experiment. It is widely used in tissue analysis of proteins as
alternative to classical histology. Application of metabolite imaging
using MS in increasing with several areas of application [104],
including lipid analysis [105]. MALDIeMS imaging to localize and
visualize molecular information in the whole worm has been also
described [82]. However, spatial resolution of available instruments
is still insufficient for use with C. elegans. First results in our lab
showed that metabolite imaging is theoretically possible, but needs
improvements in sample preparation and spatial resolution (un-
published). ToF-SIMS is an interesting alternative approach offering
very high spatial resolution. An initial study used ToF-SIMS for
analysis of C. elegans extracts, but also showed potential applica-
bility in imaging of biomolecules [83]. Future developments to-
wards higher spatial resolutions and increased sensitivity in
MALDIeMS will open new fields in the analysis of C. elegans lipids.
Knowledge transfer and standardization
Currently no centralized database for C. elegans lipid exists. The
major database for lipidomics investigations is LipidMaps, con-
taining near 40,000 unique lipid structures (status of 18.10.14)
[106]. Still, major lipids present in C. elegans, e.g. ceramides with
C17:0iso sphingoid bases or maradolipids are missing in this
database. WormBase is the current gold standard for C. elegans
biology and integration of metabolites, lipids and metabolic and
lipid pathways would greatly facilitate dispersion of knowledge.
One step in this direction was made with the creation of the SMID-
DB, storing information on C. elegans signaling molecules [107].
Current efforts in our group are directed to generate a
comprehensive in silico library of lipids potentially present in
C. elegans, which can be validated and supplemented experimen-
tally. A first step toward integration of C. elegans lipid data was done
by the LipidMaps consortium by adding lipid related genes and
proteins of C. elegans to the database.
To make results from future lipidomics experiments in the
worm comparable a standard lipid extract would be helpful. Similar
efforts have been carried out to create a NIST standard “Metabolites
in Human Plasma” [108]. Such a standard lipid extract could be run
as additional quality control to validate results and allows com-
parison of different experiments. Potentially such a standard ma-
terial can be produced for the different life stages and amount of
specific lipids can be quantified and stored in an above mentioned
database following the example of the Human Metabolome Data-
base [109,110].
Conclusion
The nematode C. elegans is capable of synthesizing a large bunch
of different lipids from fatty acids to glycerol- and glycer-
ophospholipids, steroid hormones, glycolipids and many more.
Different analytical methods have been employed to access this
large chemical diversity. C. elegans is a proven model organism for
lipid metabolism research. Lipids play a central role in the biology
of the nematode, but also in other model organisms and are often
linked intimately linked to reproduction and life span [111]. As
shown lipids, e.g. ceramides in a novel TORC pathway, can serve as
important signaling molecules. One might ask now based on this
observation: How specific is the activity of this pathway? What
lengths of the N-linked fatty acids are possible and do all of them
act in the pathway or are just a few of the active and all other are
produced transiently? The same questions can be asked for
different lipid classes. With the advent of the descriptive “omics”
technologies, including lipidomics these questions will be poten-
tially answered. Furthermore, more and more lipid standards will
become available allowing direct activity testing of them and fa-
cilitates lipid identification. A major advantage of C. elegans as
model organism is that hypothesis can be tested in a straight for-
ward manner with readily available mutants or feeding assays.
Using RNAi, specific genes can be knocked down at specific time
points without genetic mutation (knock-out), which makes it also
possible to study function of genes of which a knock-out would be
lethal during development.
These possibilities have to be combined with novel tools and
approaches already available or to be developed to fully exploit the
potential of C. elegans. However, first steps into this direction are
made.
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