Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris

Abstract

Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C15H10O5, which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.

Full text

Characterization of an anthraquinone fluor
from the bioluminescent, pelagic
polychaete Tomopteris
Warren R. Francis,
a,b
Meghan L. Powers
a,b
and Steven H. D. Haddock
a
*
ABSTRACT: Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia
when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not
been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar
spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report
the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid
chromatography-mass spectrometry revealed the mass to be 270 m/zwith a molecular formula of C
15
H
10
O
5
, which ultimately
was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor
for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. © 2014 The Authors. Luminescence
published by John Wiley & Sons Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: Tomopteris; polychaete; bioluminescence; fluorescence; anthraquinone
Introduction
The ocean is rife with luminous animals, most of which emit blue
light (1,2). An exception is the annelid worms in the genus
Tomopteris, a group of pelagic polychaetes of which several
species are reported to produce yellow bioluminescence (3).
When agitated, these animals can release glowing material into
the water that persists for several seconds. The yellow
luminescence of Tomopteris has been known for some time (4,5),
yet is unstudied when compared with bacterial, beetle or
coelenterate systems. It was reported by Harvey that homoge-
nates from the polychaete did not show a luciferin–luciferase-
type reaction nor did they produce light with the ostracod
luciferin (Cypridina luciferin), suggesting the possibility that a
previously uncharacterized luciferin is used (3). Shimomura (6)
also performed some preliminary investigations into the yellow
bioluminescence.
The connection between oxyluciferin fluorescence and the
bioluminescence has been described for several systems
including coelenterates, the firefly and luminous bacteria (1).
For coelenterates, notably Aequorea, the bioluminescence
spectrum was identical to the fluorescence spectrum of the
photoprotein following the bioluminescence reaction, that is,
coelenteramide bound by the photoprotein (7). In the case of
the firefly, the bioluminescence matches the fluorescence of
the oxyluciferin, the oxidized product of the consumable
substrate (8,9). Similarly, in bacterial systems, the biolumines-
cence spectra also matches the fluorescence of a flavin cation,
which is oxidized in the reaction and later regenerated (10–12).
With this in mind, Terio examined two fluorophores in
Tomopteris nationalis specimens, one appearing yellow–green
with ultraviolet excitation, the other yellow–orange (13,14). His
detailed observation under the microscope revealed that the
yellow–orange fluorescent material was located near the
photocytes (light-emitting cells), indicating a likely involvement
in the bioluminescence. The material had a fluorescence
emission maximum between 550 and 570 nm, and appeared
similar to the bioluminescence emission. The fluorescence was
unchanged in non-polar solvents suggesting the compound
was non-polar. Finally, Terio had speculated that this compound
might be involved in the luminous reaction, possibly as the
luciferin, but it was never characterized further. Although few
luciferins have been isolated, it is thought that bioluminescence
evolved many times and novel chemistries may still be found
(15). Fewer than 10 luciferins have been identified and the
discovery and characterization of a novel luciferin would be a
substantial advancement in the study of bioluminescence (2).
Here, we report the isolation and characterization of the
fluorescent yellow–orange material from whole Tomopterid
specimens. We were able to obtain an accurate mass of the
compound as well as the molecular formula. Through a
comparison of literature spectra and by liquid chromatograph-
mass spectrometry (LCMS), we identified the compound as
aloe-emodin, a polyhydroxyl-substituted anthraquinone. Finally,
* Correspondence to: S.H.D. Haddock, Monterey Bay Aquarium Research
Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA. E-mail:
haddock@mbari.org
a
Monterey Bay Aquarium Research Institute, Moss Landing, CA, 95039, USA
b
Department of Ocean Sciences, University of California Santa Cruz, Santa
Cruz, CA, USA
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are made.
Luminescence 2014 © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.
Research article
Received: 23 September 2013, Revised: 13 February 2014, Accepted: 20 February 2014 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2671

we speculate on possible roles based on known redox properties
and chemiluminescence from other anthraquinones.
Experimental
Samples
Tomopteris specimens were collected in Monterey Bay using
remotely-operated vehicles (ROVs) from 1999 to 2011. Many
had been caught previously and frozen in liquid nitrogen. The
specimens were found between depths of 269 and 1316 m,
typically around 400 m. Specimens varied considerably in size,
from 3 cm (∼0.5 g, wet) to >40 cm (∼50g, wet). Polychaete
taxonomists recognize that there are several undescribed
species in these waters (EV Thuesen and KJ Osborn, pers. comm.)
and all tested species had the same luminescent properties, so
no attempt was made to discern species. The condition and
amount of extractable material were also variable, due to
specimens often releasing luminous material prior to being
caught or being damaged by the sampling apparatus.
Chemicals
Water for high-performance liquid chromatography (HPLC) was
purified by reverse-osmosis. All other solvents were HPLC grade
and were purchased from Fisher Scientific. Aloe-emodin was
purchased from Sigma-Aldrich.
Extractions
Luminous material was collected when released from live
animals in a tube with gentle agitation. Frozen specimens were
homogenized using a tissue grinder. The homogenate was
divided into microfuge tubes and an equal volume of methyl ac-
etate (MeOAc) was added to each tube, typically 1 mL. This
formed emulsions. The tubes were briefly vortexed and then
centrifuged for 2 minutes at 16,000g. This separated the
emulsion into three layers: aqueous, lipids and debris, and
organic. The MeOAc layers (organic) were pooled and dried
under vacuum at ambient temperature. The sample was
reconstituted with three extractions of 20 μL MeOH and
transferred to a HPLC vial for injection.
Purification
HPLC was performed using a Shimadzu Nexera system with a
Hypersil Gold C
18
column (50 × 2.1 mm, 1.9 μm Thermo). Run
parameters were: 1 mL/min flow rate; binary gradient of H
2
O:
MeOH + 0.1% formic acid from 95:5 to 5:95 over 10 min; 60ºC
column temperature; 450 nm fluorescence excitation; 548 nm
fluorescence detection; photodiode array scans from 210 to
800 nm at 250 scans/min.
Spectra
The fluorescence and in vivo bioluminescence spectra were
acquired using a Ocean Optics QE65000 spectrometer with
attached fiber optic. The associated Ocean Optics program
SpectraSuite was used to collect spectra. The absorption spectra
were measured in a 1mL cuvette on a Tecan Infinite 200 running
Tecan i-control software. The digitized data from Terio (13,14)
were captured with ImageJ using the “Measure”and “Plot
Profile”commands to generate a graph of the intensity across
the photograph from the original papers.
Mass analysis
Low-resolution mass was determined by LCMS using a Thermo
Finnigan LC/MS/MS (LTQ) electrospray ionization (ESI) mass
spectrometer (Thermo, San Jose, CA, USA). For the LC, a Hypersil
Gold C
18
column (50 × 2.1 mm, 1.9 μm, Thermo) was used, and
run parameters were: 0.5 mL/min flow rate; binary gradient of
H
2
O: MeOH + 0.1% formic acid from 95:5 to 5:95 over 28 min;
60ºC column temperature. For the MS: negative ionization mode
(M-H); source voltage 5.0 kV; mass range from 150.0 to 1000.0 m/z;
photodiode array range from 200 to 600 nm; normalized collision
energy of 35% for MS/MS.
For accurate mass determination, the sample was dried under
vacuum and sent out to the Vincent Coates Foundation
Mass Spectrometry Laboratory at Stanford University (http://
mass-spec.stanford.edu). The sample was reconstituted in 100 μL
of 1: 1 H
2
O: MeOH and sonicated for 10 min immediately prior
to analysis.
For mass profile determination, another dried sample of the
HPLC purified compound and a standard of aloe-emodin were
sent for LCMS to the Vincent Coates Foundation Mass Spectrom-
etry Laboratory at Stanford University. The sample and standard
were reconstituted in 50 μL 1:1 H
2
O: MeOH, vortexed for 30 s
then sonicated for 10 min. A portion was diluted 1:10 with
H
2
O:MeOH and transferred into an HPLC vial. For the LC, a
Agilent C
18
column (50 × 2.1 mm, 1.8 μm) was used, with
parameters: 0.2 mL/min flow rate; binary gradient of H
2
O:
acetonitrile + 0.1% formic acid from 90:10 to 0:100 over 10 min.
The mass was analyzed with a Bruker MicroTOF-QII quadrupole
time of flight mass spectrometer in negative ESI mode.
Results
Acquisition of raw material
Specimens were caught at depth using ROVs. This often enabled
careful capture of very large specimens that could be returned
to the lab in excellent condition. When agitated, luminescence
begins in the parapodia and nearly all of our specimens released
glowing material from their parapodia (Fig. 1B). To our
knowledge, there is no mention in the literature of these animals
releasing luminescent particles. We consider that this may due
to the majority, possibly all, of the specimens in the literature
being agitated or injured during capture using plankton nets.
We acquired the bioluminescence spectra of the luminous
exudate (shown in Fig. 2A, λ
max
: 565 nm), which is in good
agreement with the Atlantic species Tomopteris nisseni
measured by Latz (16). The bioluminescence spectrum also
matches perfectly with the digitized fluorescence spectrum of
the yellow–orange fluor measured by Terio (14), to the extent
that the image may be converted to a spectrum.
Because live specimens release glowing material, we reasoned
that the light emitter could be isolated from the exudate.
Luminous exudate has a bright yellow–orange fluorescence
under blue light, however, the quantity obtained was insufficient
for further analysis. Whole animals displayed a bright yellow–
green fluorescence around the coelom in the parapodia even
when fixed or frozen (Fig. S1A). This material was clearly visible
as a bright yellow pigment in the parapodia for frozen
W. R. Francis et al.
Luminescence 2014wileyonlinelibrary.com/journal/luminescence © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.

specimens and was seen even in specimens frozen for over
10 years. Owing to the irregularity of acquiring new specimens
at sea and collecting exudate, we instead extracted material
from frozen specimens (see Experimental).
Non-polar extractions
Frozen specimens were homogenized, and MeOAc was added to
the homogenate. After centrifugation, nearly all of the
fluorescent material was in the non-polar phase and appeared
pale yellow. The absorption spectrum of the non-polar phase
showed a large peak at 364 nm (Fig. S2). The aqueous layer was
dimly fluorescent green, likely due to riboflavin or a similar com-
pound. Often, the fluorescence of the MeOAc layer appeared
bright yellow–green immediately after extraction (λ
max
: 519 nm).
When exposed to blue light, this changed to the characteristic yel-
low–orange color in seconds (Fig. S1B). This effect was attenuated
in the presence of ascorbic acid, suggesting that oxygen or reac-
tive oxygen species could be involved in this transition.
Purification of the yellow-orange compound by HPLC
This crude organic extract was separated by reversed-phase HPLC
to isolate the fluorescent yellow–orange pigment (Fig. 3A). Very
large absorption peaks at 254 and 430 nm of a yellow material
with a bright fluorescence peak were observed around 5.6 min
(Fig. 3B). This single peak was collected over multiple injections.
The absorption (λ
max
: 286, 430nm) and fluorescence emission
(λ
max
: 580, 548 nm shoulder) were acquired for the purified com-
pound (Fig. 2B). The absorption peak of the purified compound is
430 nm, however, this does not appear to be abundant enough in
the unpurified extract to show a distinct peak (Fig. S2). Instead, it
likely that some other pigment accounts for the peak at 364 nm in
the original MeOAc extract. Although the fluorescence emission
Figure 1.Tomopteris bioluminescence. (A) Photograph of a typical specimen, taken by the camera on the ROV. (B) Photo of the yellow bioluminescence, which begins at
the tips of the parapodia and is then released into the water. The animal is oriented with the head at the bottom-left with the body axis up to the top-right. The camera was
a NIKON D3 with ISO of 6400 and 10 s exposure time. Exposure time was longer than the luminescent emission to ensure capturing the event. Color is the natural color of
luminescence as captured by the camera. (C) Chemical structure of aloe-emodin.
Figure 2. Various spectra. (A) The in vivo bioluminescence spectra of Tomopteris (solid line) and digitized in vivo fluorescence data from Terio (14) (dotted line). (B) Absorption
spectra of the fluorescent pigment in methanol (dashed line) and with a drop of NaOH (dotted line), as well as the fluorescence emission spectrum in chloroform (solid line).
Afluor from the bioluminescent polychaete Tomopteris
wileyonlinelibrary.com/journal/luminescenceLuminescence 2014 © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.

does not perfectly match the digitized spectrum reported by Terio
or the bioluminescence (Fig. 2B), (14) this may be due to the
solvent or that the spectrum changes when bound by a protein,
as seen for coelenterazine (17–19).
Mass determination and molecular formula
Knowing the absorption spectrum of the compound permitted
easy mass determination of the compound with LCMS. The same
MeOAc extract was analyzed by LCMS, where the yellow
compound was identified at 337 m/zwith the major fragment
at 269 m/z(Fig. S3), corresponding to a mass difference of
68 m/z.Tofind the molecular formula and the identities of the
fragments, the accurate mass was determined for the purified
compound at 337.0331 m/z, corresponding to C
15
H
9
O
5
+
NaCHO
2
(M-H). It was then determined that the major fragment
was actually the molecular ion, at 269.0455 m/z, which indicated
loss of the sodium formate adduct and the uncharged molecular
formula of C
15
H
10
O
5
(Figs S4–S6).
Confirmation of the identity as aloe-emodin
The fluorescent material in methanol undergoes a bathochromic
shift from yellow to red upon addition of saturated NaOH
solution (Fig. 2B, dotted line, λ
max
: 510 nm, also in Fig. 4).
The spectra and this transition are thought to be a property of
1,8-dihydroxy-9,10-anthraquinones (20). After comparison of
our spectrum with 20 published UV/vis spectra of anthraqui-
nones with the same molecular formula (21,22), we noticed that
our spectrum is remarkably close to the reported spectrum of
aloe-emodin (structure in Fig. 1C) (23). Aloe-emodin was
purchased (Sigma-Aldrich) and was found to have an identical
absorption spectrum as the yellow–orange fluor (Fig. 4).
The product ion mass spectrum (MS-MS) is sometimes used to
confirm the presence of rare metabolites for cases where NMR
cannot be used to deduce the structure (24). To ultimately
confirm the identity of the compound, the HPLC-purified sample
and a standard of aloe-emodin were sent out for analysis by
LCMS. The retention time, the calculated and measured m/z
ratios, and the product ion spectra were all identical matches,
consistent with the hypothesis that the yellow–orange fluor is
indeed aloe-emodin (Figs S7–S9).
Discussion
Extraction yield
Here, we described the extraction and identification of the yellow–
orange fluor in the Tomopteris, which was first noted over 50 years
ago. As the mass and structure were only determined towards the
end of our experiments, some questions related to extraction
yields were unaddressed. However, estimated from the published
extinction coefficients of aloe-emodin, the HPLC data (from Fig. 3)
suggest that the single injection of 10 μL contains in the order of
35 μg of aloe-emodin. Because multiple HPLC runs were necessary
to separate all the material and not saturate the column, we esti-
mate that even a relatively small worm (3–5 cm, estimated to be
200–500 mg) could contain 200 μg of aloe-emodin. Measurements
of other Tomopteris specimens suggest dry material accounts for
around 15% of the mass (25). For a 500 mg worm, this means that
dry mass accounts for 75 mg, where 200 μg of aloe-emodin is al-
most a third of a percent of the dry mass.
Functions of quinones
We have ultimately confirmed the compound to be aloe-
emodin, but we do not know the function of aloe-emodin for
this marine animal. Given that aloe-emodin is an anthraquinone,
it is logical that it is used similarly as other anthraquinones.
There are a number of cases for insects in which quinones and
anthraquinones have been suggested to have various defensive
Figure 3. HPLC chromatogram of the MeOAc extract. (A) The UV/vis absorption (254 and 430 nm) and fluorescence chromatograms show a large peak of the fluorescent
yellow–orange compound at 5.6 min, indicated by the star. (B) The corresponding absorption spectrum at 5.6 min clearly showing the characteristic peak at 430 nm.
Figure 4. Spectra of aloe-emodin and the yellow–orange fluor. The measured
absorption spectra of the purchased aloe-emodin and the yellow–orange fluor in
methanol and with a drop of saturated NaOH solution.
W. R. Francis et al.
Luminescence 2014wileyonlinelibrary.com/journal/luminescence © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.

roles, possibly as toxins (20,26,27). Quinones also are known to
participate in redox reactions, such as in the electron transport
chain. Because all known bioluminescence reactions involve an
oxidation (15), quinones are well suited for this type of
chemistry. Aloe-emodin has been discussed in literature for both
antioxidant and prooxidant properties, making a strong case for
its role in this regard (28–30).
Quinones in other bioluminescent systems
Furthermore, there is a precedent of a quinone in biolumines-
cence from an unusual polybrominated benzoquinone that is
used in the luminous system of the acorn worm, Ptychodera
flava, which also requires riboflavin (31,32). Given that the green
color of the light of the acorn worm closely matches the
fluorescence of riboflavin, it is possible that riboflavin is the light
emitter and this benzoquinone serves as an electron carrier for
the oxidation of riboflavin. Alternatively, the authors of that work
had demonstrated that polybrominated quinones themselves
were chemiluminescent, suggesting that perhaps riboflavin is
only present as a fluor for resonant energy transfer to change
the color of the emitted light.
Chemiluminescence of anthraquinones
Other anthraquinones have been shown to be chemilumines-
cent (λ
max
: 568 nm) when reduced to the hydroquinone or
semiquinone and reacted with molecular oxygen (33). Addition-
ally, it was also shown that a semiquinone form was chemilumi-
nescent (or fluorescent) in yellow–green (λ
max
: 515 nm) (33). We
hypothesize that aloe-emodin, a substituted anthraquinone,
would have very similar properties. In fact, our observations of
afluorescent yellow–green compound which transitions to
aloe-emodin (where it is fluorescent yellow–orange) suggest
the possibility that the yellow–green compound is a reduced
form of aloe-emodin, possibly the anthrone which would be
very susceptible to oxidation (34,35). If aloe-emodin were the
oxyluciferin in this context, then plausibly the fluorescent
yellow–green compound, the anthrone or a similar compound,
could be the luciferin.
Other discussions of Tomopteris
The only modern characterization of Tomopteris luminescence
suggested that chemiluminescence could be elicited from
homogenate with superoxide ions (6), as seen for several other
polychaetes (36,37). A large amount of Triton-X (2%) was needed
to solubilize the light-emitter, suggesting that the enzyme may
be a membrane-bound photoprotein (6). However, we consider
it is unlikely that the in vivo mechanism of light emission
requires superoxide. For example, coelenterazine is chemilumi-
nescent with superoxide yet the light output was an order of
magnitude lower than the same quantity of coelenterazine
bound to obelin and activated with calcium ions (38).
Theories of origins of aloe-emodin
It was surprising to find this compound in a deep-sea animal as it
was discovered from several Aloe species. It is not known
whether the Tomopteris synthesizes aloe-emodin or acquires it
elsewhere, perhaps through its diet or from a symbiont. Many
anthraquinones are biosynthesized through a convergent
mechanism using polyketide synthases (39,40), a mechanism
that is conserved across prokaryotes, fungi and plants, thus
any of those modes of acquisition may be possible. A dietary link
from land plants would be preposterous; however, there are
other cases of anthraquinones from marine organisms, (41)
including a marine fungus that lives commensally with a green
alga and appears to produce several anthraquinones and an
isomer of aloe-emodin (42). Another possibility is that a
symbiont is generating the compound and there is some
precedent for this scenario in metazoans. It was thought that
some insects may synthesize their own polyketides (40),
although one study had shown that the compounds were made
by an uncultured bacterial symbiont (43). To our knowledge,
there has not been a confirmed case of polyketide synthesis
by metazoans. Although this does not rule out such a possibility,
it suggests that the aloe-emodin from Tomopteris may ultimately
derive from another organism or involve biosynthetic mecha-
nisms other than polyketide synthases.
Conclusions
From our detailed purification and LCMS, we have shown that
the fluorescent yellow–orange compound in Tomopteris is
aloe-emodin. Evidence from the overlap of the fluorescence
and bioluminescence spectra is very compelling to suggest that
aloe-emodin is the final light-emitter for Tomopteris biolumines-
cence. While evidence from related systems favors the interpre-
tation that aloe-emodin is the oxyluciferin, this does not exclude
the possibility that aloe-emodin is an acceptor for resonant en-
ergy transfer from another molecule. Detailed chemical studies
are needed to discern these two cases. Ultimately, full character-
ization of the Tomopteris luminous system may lead to a new
generation of bioluminescent sensors or reporters, particularly
for plants or fungi where many anthraquinones are endogenous.
Authors’contributions
WRF and SHDH designed experiments and analyzed data. WRF,
MLP and SHDH caught animals. MLP and SHDH acquired the
bioluminescence spectrum. WRF did the experiments. WRF
wrote the paper with corrections from the other authors.
Acknowledgements
WRF would like to thank R Linington for helpful discussions and
advice. The NIH National Institute of General Medical Sciences
(ROI-GMO87198) to SHDH supported our work. This research
was also supported by the David and Lucile Packard Foundation
through the Monterey Bay Aquarium Research Institute.
University of California-Santa Cruz LCMS facility was funded by
NIH grant S10RR020939.
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Supporting information
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version of this article at the publisher’s web site.
W. R. Francis et al.
Luminescence 2014wileyonlinelibrary.com/journal/luminescence © 2014 The Authors. Luminescence published by John Wiley & Sons Ltd.