Long-chain O-ascarosyl-alkanediols are constitutive components of Caenorhabditis elegans but do not induce dauer larva formation

Article abstract of DOI:10.1002/cbdv.201000012

Two long-chain ascarosides, O-ascarosylnonacosane-2,28-diol (1) and O-ascarosyluntriacontane-2,30-diol (2), were isolated from Caenorhabditis elegans and detected in all developmental stages of the worm. The long-chain ascarosides were shown to be minor lipid components, and it was also shown that they do not induce dauer larva formation.

Full text of DOI:10.1002/cbdv.201000012

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Long-Chain O-Ascarosyl-alkanediols Are Constitutive Components of
Caenorhabditis elegans but Do Not Induce Dauer Larva Formation
by Vyacheslav Zagoriy*, Vitaliy Matyash, and Teymuras Kurzchalia
Max-Planck Institute for Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden
(phone: þ49-351-2102633; e-mail: zagoriy@mpi-cbg.de)
Two long-chain ascarosides, O-ascarosylnonacosane-2,28-diol (1) and O-ascarosyluntriacontane-
2,30-diol (2), were isolated from Caenorhabditis elegans and detected in all developmental stages of the
worm. The long-chain ascarosides were shown to be minor lipid components, and it was also shown that
they do not induce dauer larva formation.
Introduction. – Ascarosides, glycolipids containing the 3,6-dideoxysugar ascarylose
(3,6-dideoxy-l-arabino-hexose), have been described in parasitic nematodes of the
genus Ascaris, almost a century ago [1][2]. Based on the structure of the side chain,
they are classified as long-chain monool glycosides, diol monoglycosides and diol
diglycosides [3]. Ascarosides of Ascaris are present in embryos, mainly in the form of
esters of acetic, propylic, and isovaleric acids [2]. Upon fertilization, the ascarosides are
deesterified and translocated to the inner layer of the newly formed eggshell, being a
major constituent of this layer and providing eggshell impermeability [4]. Ascarosyl
compounds were also described in Caenorhabditis elegans, where they are dauer larva-
inducing and sexual pheromones [5]. The aglycones of these C. elegans ascarosides are
short-chain alkanes with COO and C¼O groups [6][7]. Recently, it has been reported
that long-chain O-ascarosyl-(2,wꢀ1) alkanediols with 29 and 31 C-atoms were present
in growth media of this nematode [8]. In this study, we addressed the question whether
long-chain ascarosides are present in different life-stages of the worm, and investigated
their ability to induce dauer larva formation.
Results and Discussion. – First, we tried to answer the question whether long-chain
ascarosides are present in the developmental stages of C. elegans. For this purpose, a
lipid extract of C. elegans mixed populations was prepared according to Bartley et al. [3]
with modifications. After saponification, the hydrophobic compounds were extracted
with CHCl3 and separated by RP-HPLC/MS, to check for the presence of molecular
weights corresponding to long-chain ascarosides, previously described in different
Ascaris species. Molecular ions potentially corresponding to O-ascarosylnonacosane-
2,28-diol (1; m/z 569.6 ([MꢀH]ꢀ )) and O-ascarosyluntriacontane-2,30-diol (2; m/z
597.6 ([MꢀH]ꢀ )) were observed.
To confirm that these molecular ions correspond to long-chain ascarosides, we have
isolated them from the saponified lipid extract of C. elegans, using preparative 2D thin
layer chromatography (TLC). After visualization of the control TLC plate, corre-
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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sponding regions of the preparative plates were back-extracted and checked for the
presence of long-chain ascarosides with 2D HPTLC and HPLC/MS, using long-chain
O-ascarosyl alkanediols isolated from Ascaris suum as a reference. The TLC behavior
of the long-chain ascarosides from C. elegans corresponded to that of the reference,
with Rf 0.3 in 1D and 0.1 in 2D TLC, respectively (Fig. 1,a und c). When the mixture
of ascarosides from C. elegans was analyzed by HPLC/MS (gradient elution, see
Exper. Part), the peaks of 1 and 2 were observed at m/z 569.6 ([MꢀH]ꢀ ) and 597.6
([MꢀH]ꢀ ), respectively (Fig. 1,b). The chromatographic behavior of 2 (tR 22.0 min)
isolated from C. elegans corresponded to that of 2 isolated from A. suum (Fig. 1,b and
d). As expected, from A. suum, also O-ascarosyltritriacontane-2,32-diol (3) and
O-ascarosylpentatriacontane-2,34-diol (4) were isolated. The differences between the
tR of compounds 2, 3, and 4 isolated from A. suum were the same as that between
compounds 1 (tR 20.0 min) and 2 isolated from C. elegans. This confirmed the
assumption that 1 is O-ascarosylnonacosane-2,28-diol.
To definitively establish the identity of the sugar residue, long-chain ascarosides of
C. elegans were subjected to acidic hydrolysis, yielding ascarylose, as determined by
HPLC/MS (tR 2.3 min; ESI-MS (negative-ion mode): m/z 207 ([MþCH3COO]ꢀ )),
using synthetic ascarylose as a reference (Fig. 2). When performing MS/MS fragmen-
tation of parent masses of 1 and 2, neutral losses of sugar (130 Da) and two H2O
residues (147 and 164 Da) were observed, confirming their identity. Thus, in C. elegans,
diol monoglycosides 1 and 2 constituted the major types of long-chain ascarosides.
It is known that long-chain ascarosides in Ascaris are esterified. Therefore, the
question was addressed, whether the same is true for C. elegans. The concentrations of 1
and 2 were compared in the equivalent lipid extracts of adult worms before and after
saponification, using the same HPLC/MS system as for the detection of the ascarosides.
The amounts of 1 and 2 were ca. eight times higher after saponification (P<0.05).
Thus, in C. elegans, ascarosides are mainly present as esters. Their presence in the
culture media in the unesterified state [8] might be due to the release by enzymatic
hydrolysis, similar to the way it occurs during the formation of the eggshell in Ascaris
[4].
Moreover, it was investigated whether the ascarosides are constitutive components
during the life cycle or whether they accumulate at a certain developmental stage. The
relative amounts of ascarosides in the different life stages were measured by HPLC/MS,
using the same system as for their detection, and the amounts were normalized either to
the number of worms (Fig. 3,a) or to the protein content (Fig. 3,b). The amount of
ascarosides per worm steadily increased, reaching a maximum at the adult stage.

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Fig. 1. a) 2D TLC of ascarosides isolated from C. elegans. b) HPLC/ESI-MS (negative-ion mode) of
ascarosides isolated from C. elegans. Selected ion traces: m/z 569 (1), 597 (2), and 625 (3). The ion at m/z
653 (4) was not detected. c) 2D TLC of ascarosides isolated from A. suum. Fatty acids were also observed
(Rf 0.5 and 0.2 in 1D and 2D TLC, resp.). d) HPLC/ESI-MS (negative-ion mode) of ascarosides isolated
from A. suum. Selected ion traces: m/z 597 (2), 625 (3), and 653 (4). The ion at m/z 569 (1) was not
detected.
Absolute quantification of long-chain ascarosides was performed by HPLC/MS,
using long-chain ascarosides isolated from A. suum as a standard. To achieve better
linearity, an isocratic elution system was used. The area under the peak of known
amounts of 3 was used to establish the calibration curve. Analyzing the ascarosides
from synchronized populations of C. elegans, the amount of ascarosides was estimated
to be ca. 150 pg per adult worm. When compared to the amount of phospholipids at the
same developmental stage (ca. 15 ng per adult worm), long-chain ascarosides
constitute ca. 1%. This is in contrast with what was observed in parasitic nematodes,
where the ascaroside esters constituted up to 33% of all neutral lipids in the ovaries [9].
Interestingly, when normalized to the protein content, the amount of long-chain
ascarosides reached its maximum at the L2 stage, thus indicating that they might be
involved in the regulation of dauer larva formation, dauer larva being an alternative to

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Fig. 2. HPLC/ESI-MS (negative-ion mode; mass chromatogram of m/z 207) of the sugar residue
obtained after acid hydrolysis of long-chain ascarosides isolated from C. elegans. The tR of ascarylose and
the reference carbohydrates in this system are indicated with arrows.
L3 under unfavorable conditions. To clarify whether long-chain ascarosides induce
dauer formation similar to short-chain ascarosides (daumones) [5], the dauer-inducing
activity was checked using the diol monoglycosidic ascarosides isolated from female A.
suum with a recently described assay [10]. Long-chain ascarosides did not show any
dauer-inducing effect at similar or higher concentrations than those of short-chain
ascarosides (Fig. 3,c). These data show, that compounds 1 and 2 do not seem to be
signaling molecules, and it is unlikely, that they are precursors for daumones [11].
Thus, long-chain ascarosides are present in C. elegans in all stages of development.
Although their function still has to be elucidated, long-chain ascarosides do not seem to
be involved in eggshell formation or dauer larva induction.
We would like to thank Prof. H.-J. Knoelker (Dresden, Germany) for providing synthetic ascarylose,
Dr. D. Schwudke for help with MS/MS experiments, and the members of the lab of T. K. for thoughtful
discussions.
Experimental Part
General. All chemicals were purchased from Sigma-Aldrich (Germany), unless otherwise stated.
Ascarylose was provided by the Department of Chemistry, TU Dresden (Organic Chemistry; Head, Prof.
H.-J. Knoelker); the synthesis is described in [12].
Animal Material. Caenorhabditis elegans. Liquid cultures of mixed population N2 worms were
grown according to Wood [13]. Homogenous populations were obtained as follows. Embryos of the wild-
type Bristol N2 strain were obtained by treating the mixed population with 4% aq. NaOH soln. in
household bleach (3–6% aq. NaClO), and were suspended in M9 buffer for 48 h at 208. Produced L1

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 3. Relative abundance of ascarosides in the developmental stages of C. elegans normalized to a) the
number of animals and b) the protein content. c) Effect of long-chain ascarosides isolated from C. elegans
on the induction of dauer larva formation. Data are presented as meansꢁSD.
were transferred to nematode growth medium (NGM) agar covered with bacteria, and the plates were
incubated at 208. The worms were washed off with M9 buffer after 10, 20, 30, or 40 h of incubation, to
obtain L2, L3, L4, or young adult homogenous populations, resp. Dauer larva were obtained using a daf-7
mutant strain (CGC, UK) grown at 258.

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Ascaris suum. Ascaris suum were obtained from the Zoology Department of the TU Dresden (Head,
R. Entzeroth). Females were dissected to excise the uterus, which was used for further procedures.
Sample Preparation. All samples were homogenized in a minimal amount of H2O, and the extraction
and sample pretreatments were performed according to Matyash et al. [14]. Before the extraction, an
aliquot of d-glucosyl-b-1,1’-N-lauroyl-d-erythro-sphingosine (AvantiLipids) was added, to account for
the treatment losses.
TLC. Prep. TLC was performed on silica-gel 60 HPTLC plates (20ꢂ20 cm; Merck) developed with
CHCl3/MeOH 9 :1 (first dimension) and CHCl3/MeOH 24 :1 (second dimension). Anal. 2D TLC was
performed in the same way, using silica-gel 60 TLC plates (10ꢂ10 cm; Merck). Bands were visualized by
spraying the plates with 20% H2SO4 in EtOH and charring at 2008. For prep. purposes, the silica-gel was
scraped with a scalpel, extracted with a tenfold amount of MeOH, and filtered. Initial solns. (10 ml) of
packed C. elegans worms yielded ca. 0.5 mg of long-chain ascarosides.
O-Ascarosylnonacosane-2,28-diol (1). tR (System 2) 23.5 min. ESI-MS/MS (positive-ion mode) of
m/z 588.427 ([MþNH4]þ ): 405.443 ([MþHꢀ2 H2OꢀC6H10O3]þ ), 423.44 ([MþHꢀH2OꢀC6H10O3]þ ),
441.461 ([MþHꢀC6H10O3]þ ).
O-Ascarosyluntriacontane-2,30-diol (2). tR (System 2) 29.5 min. ESI-MS/MS (positive-ion mode) of
m/z 616.456 ([MþNH4]þ ): 433.472 ([MþHꢀ2 H2OꢀC6H10O3]þ ), 451.469 ([MþHꢀH2Oꢀ
C6H10O3]þ ), 469.490 ([MþHꢀC6H10O3]þ ).
O-Ascarosyltritriacontane-2,32-diol (3). tR (System 2) 38.2 min.
HPLC. All HPLC separations were performed with a Waters Alliance 2695 pump coupled to TOF
MS. System 1. The detection and relative quantification of ascarosides was performed with an Agilent
XDB C8 RP column (4.6ꢂ150 mm, 5 mm) and gradient elution with 100% Solvent A (MeOH/0.1% aq.
ammonium acetate 9 :1) for 5 min, 0% Solvent B (MeOH/0.1% aq. ammonium acetate 99 :1) to 100% B
in 5 min, and 100% B for 35 min, followed by column equilibration with 100% A for 15 min. System 2.
The absolute quantification of ascarosides was performed with an Agilent XDB C8 reversed-phase
column (3.0ꢂ150 mm, 3.5 mm,) eluted with MeOH/0.1% aq. ammonium acetate 9 :1 for 45 min. For
both systems, the flow rate was 0.3 ml/min, the column temp. was maintained at 408, and the injection
volume was 10 ml.
HPLC Separation of Carbohydrates. Carbohydrates were analyzed using an Agilent aminopropyl RP
column (2.6ꢂ150 mm, 5 mm); elution was isocratic with MeCN/0.1% aq. ammonium acetate 85 :15 for
20 min; the flow rate was 0.15 ml/min.
MS Analysis. MS Analysis was carried out on an LCT TOF mass spectrometer (Waters Inc.) with an
ESI source. Instrument operation and data acquisition was carried out using MassLynx 4.1 software. N2
was used as the cone and nebulizing gas. The ionization voltage was set to 3.0 kV. Acquisition was
performed over the m/z range 100–1000. Tandem MS analysis was performed with a modified QSTAR
Pulsar i quadrupole TOF apparatus (MDS Sciex) equipped with a NanoMate HD ion source (Advion
BioSciences). The ionization voltage was set to 1.05 kV. The anal. quadrupole Q1 was operated under the
unit resolution settings, and fragments of preselected masses were detected within the m/z range 100–
1,000.
Phosphate Assay. The phosphate assay was performed according to [15].
Protein Qunatification. The amount of protein was determined with a Micro BCA protein assay kit
(Pierce).
Acidic Hydrolysis. Acidic hydrolysis was performed according to [16].
Saponification. For the saponification, the dry sample was dissolved in a ten-fold amount of 20n aq.
KOH soln./H2O 1:19, and the mixture was incubated for 1 h at 658. Then, the phase separation was
induced by adding H2O/CHCl3 1:2. The lower phase was collected and dried in a vacuum centrifuge. The
dry extract was used for further procedures.
Dauer Larva Induction Assay. The dauer larva induction assay was performed according to [9], with
monoascarosyl alkanediol (25 or 250 mg per well) isolated from A. suum.
Statistical Analysis. Data are presented as means with standard deviation. The results were
statistically analyzed by one-way analysis of variance (ANOVA), followed by a Studentꢂs t-test (P
values<0.05 were considered significant).

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Received January 15, 2010