Long-Wave Sensitivity in Deep-Sea Stomiid Dragonfish with Far-Red Bioluminescence:

Evidence for a Dietary Origin of the Chlorophyll-Derived Retinal Photosensitizer of

Malacosteus niger
Author(s): R. H. Douglas, C. W. Mullineaux and J. C. Partridge
Source: Philosophical Transactions: Biological Sciences, Vol. 355, No. 1401, Sensory Processing
of the Aquatic Environment (Sep. 29, 2000), pp. 1269-1272
Published by: The Royal Society
Stable URL: http://www.jstor.org/stable/3066659 .
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B
9;

doi

THE ROYAL
SOCIETY

10.1098/rstb.2000.0681

Long-wavesensitivityin deep-sea stomiid

dragonfishwithfar-redbioluminescence:
evidence fora dietaryoriginof the
chlorophyll-derived
retinalphotosensitizer
of Malacosteus niger
R. H. Douglasl*,C. W. Mllllineaux2and J. C. Partridge3
Applied
Vision
Research
Centre,
Department
ofOftometry
andVisualScience,
CityUniversity,
311-321Goswell
Road,
London
EC1V7DD, UK
2Department
ofBiology,
University
College
London,
DarwinBuilding,
Gower
Street,
London
WC1E6BI; UK
3School
ofBiological
Sciences,
University
ofBristol,
Woodland
Road,Bristol
BS8 I UG,UK
Bothresidualdownwelling
sunlightand bioluminescence,
whichare thetwomainsourcesofillumination
availablein the deep sea, have limitedwavebandsconcentrated
around450-500 nm. Consequently,
the
wavelengths
of maximumabsorption
of the vast majorityof deep-sea fishvisual pigmentsalso
clusterin this part of the spectrum.Three genera of deep-sea loosejawed dragonfish(Aristostomias,
Pachystomias
and Malacosteus),
however,in additionto theblue bioluminescence
typicalof mostdeep-sea
animals,also producefar-redlight (maximumemission >700nm) fromsuborbitalphotophores.
All
threegeneraare sensitivein thispart of the spectrum,to whichall otheranimalsof the deep sea are
blind,potentially
affiording
thema privatewavebandforilluminating
preyand forinterspecific
communicationthatis immunefromdetectionby predators
and prey.Aristostomias
and Pachystomias
enhancetheir
long-wavevisual sensitivity
by thepossessionofat leastthreevisualpigmentsthatare long-waveshifted
valuesca. 515,550 and 590nm) comparedwiththoseof otherdeep-seafishes.Malacosteus,
on the
otherhand,althoughit does possesstwo ofthesered-shifted
pigments
valuesca. 520 and 540 nm),
lacksthemostlong-wave-sensitive
pigmentsfoundin theothertwogenera.However,it further
enhances
itslong-wavesensitivity
witha chlorophyll-derived
photosensitizer
withinitsoutersegments.
The fluorescence emissionand excitationspectra of this pigmentare very similar to spectra obtained from
mesopelagiccopepods,whichare an importantcomponentof diet of Malacosteus,
suggesting
a dietary
Orlglntort]1S plgmeIlt.
(SImaX)

(SImaX

(SImaX

Keywords: fish;visualpigment;bioluminescence;
photosensitizer;
chlorophyll;
deep sea
The deep sea is by far the largesthabitaton Earth, shallowerdepths.Yetmanyanimals,includingmostfishes,
coveringover 60% of its surfaceand havingan average living well below the reach of sunlight,have wellThis allows themto view the
depthclose to 4000m. Despite this,less is knownabout developedvisual systems.
it than about mostotherenvironments.
However,it has second sourceof lightin the deep sea: bioluminescence
long attractedthe attentionof visionscientists
who have producedby over 80% of the species inhabitingthis
are
shownthatthe eyes of deep-sea fishesdisplayan aston- region.Both residualsunlightand bioluminescence
veryrestricted
withmostradiationbeingin the
ishingdiversity
ofanatomicaladaptationsto theirunique spectrally
therefore,
the vast
environment
(Locket 1977;Wagneret al. 1998).The deep region450-500nm. Not surprisingly,
sea has also lured those with an interestin visual majorityof fisheshave visual pigmentswithpeak absorin thisregionof the spectrum,providinga
pigmentecology.One of the aims of such work is to bance
good matchbetweenenvironment
and visual pigments,
relate the spectral absorption characteristicsof an
although detailed analysis shows that even here the
animal'svisualpigmentsto itsspectralenvironment.
The
is not straightforward
(Partridgeetal. 1989;
deep sea holdsperhapsa specialattraction
forsuchwork relationship
as, unlikein shallowwateror on land, its photicenvir- Douglas etal. 1998a).
Althoughthewavelength
ofmaximumemissionofmost
onmentis in many aspects quite simple.Thus, some
occursin theblue regionofthe
residualsunlightcan penetratethe upper 1000m of the deep-seabioluminescence
ocean in ideal conditions,although in most waters spectrum,thisis not alwaysso. The moststrikingexcep(Malacosteus,
visiblesunlightis effiectively
extinguished
at significantly tionsare threegeneraofdeep-seadragonfish
Aristostomias
and Pachystomias;
orderStomiiformes,
family
*Author
forcorrespondence
(r.h.douglas(city.ac.uk).
Stomiidae),which,in additionto blue bioluminescence
(SImaX)

Phil.Wrans.
R. Soc.Lond.B (2000) 355, 1269-1272

1269

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C) 2000 The Royal Society

1270 R. H. Douglasand others Dietary

origin
ofretinalphotosensitizer

(a)

1.0

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t\,
\
\
\
\
X

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o
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(b)

'e

* -

ut
ut

.:

* -

'e

tt
tt
t
t

'

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0.0

400

500

--t'-

600
wavelent,th
(nm)

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700

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,
800

Figure1. (a) Bioluminescence

ofAristostomiaJ
stitzmanni
(dashed
line;Widderetal. 1984),andthebest-fit
templates
(solid
lines)ofthethreevisualpigments
so faridenl
tified
initsretina
(a rhodopsin-porphyropsin
pigment
pairwit]
hilmaX values520
and551nmanda rhodopsin
witha
at 5<
& Douglas1995). The lighter
linerepresents
a theoretlcal
of
porphyropsin
with at 669nm,whichis t]he'partner'
thelong-wave-sensitive
rhodopsin
(calculatec
dusing
a formula
ina
basedonempirical
datarelating values4Ofpigments
rDouglas
etal.
rhodopsin-porphyropsin
pigment
pair)(afte]
1998a). (b) Bioluminescence
ofM. niger
(dash
ledline;Widder
etal. 1984),andbest-fit
templates
(solidlines) ofthetwo
identified
visualpigments
(a rhodopsin
withimax515nmand
a porphyropsin
with
540nm).The absot
rption
spectrum
represented
by
oftheputative
photosensitizer
(lighter
line)ivs
a purified
diethyl
etherextract
ofa retinal
suspension
(after
Douglasetal.1999).
ilmaX

SImaX

ilmaX

Slmax

similarto thatofotherdeep-seaanimals,alsohavesuborwith
bitalphotophores
producingfar-red
bioluLminescence
spectralemissionspeakingsharplyat wasselengths
beyond
700 nm (Dentonet al. 1970,1985;Widder#
etal. 1984).
We,and others,have shown,usingconIventional
retinal
extractspectrophotometry,
thatto facilitX
ateperception
of
members
of all
theirown long-wavebioluminescence,
threegenerahave at least two visual piigments
thatare
long-waveshiftedcomparedwiththosecfotherdeep-sea
etal.
animals (SImaX ca. 515 and 550nm) (e.g.. Partridge
1989; Bowmakeret al. 1988; Partridgezk Douglas1995;
forma
Douglas et al. 1998b, 1999). Such piLgments
'rhodopsin-porphyropsin
pigmentpair'' based on the
same opsin,whichin some photorecept(
orsis boundto
retinaland in othersto 3,4-dehydroreti:
nal.

Although
suchpigments
area better
match
tothefar-red
bioluminescence
oftheseanimalsthanthevisualpigments
ofmostdeep-seaanimalsthathaveima.Y
valuesaround
480-490 nm,thematch
between
pigments
absorbing
optimally at 515-550
nm, and bioluminescent
emissions
peakingabove700nm,is stillfarfromperfect.
Usinga
retinalwhole-mount
technique,
however,
whichisolates
pigments
thatdonotsurvive
retinal
extraction
or-any
form
ofpreservation
(Douglasetal. 1995),wehavebeenableto
showthatbothAristostomias
tittmanni
(Partridge
& Douglas
1995) and Pachystomias
microdon
(Douglaset al. 1998a)
possessa thirdpigment
withSImaX around588-595nm
(figure
la).Thesepigments,
whicharebysomemargin
the
mostlong-wave-sensitive
rod pigments
everdescribed,
appear,basedon theshapeoftheirabsorption
spectrum,
to use retinalas theirchromophore
boundto a second
long-wave
opsin.Itwouldtherefore
notbe unreasonable
to
supposethattheseretinas
might
actually
contain
a fourth
visualpigment
composed
of3,4-dehydroretinal
boundto
this long-waveopsin.Such a pigmentwouldgive a
virtually
perfect
matchtotheseanimals'bioluminescence
(figure
la). Unfortunately
wewereunabletoisolatesucha
pigment,
probably
becausetissuepreparations
weremade
underdimredillumination,
which
wouldcausesuchlongwave-sensitive
pigments
tobleach.
ThusbothAristostomias
and Pachystomias
containthree,
andpossibly
four,
verylong-wave-shifted
visualpigments
allowingthemto see theirownbioluminescence,
which
wouldbe invisible
to all otherspecies.Such a system
potentially
enablesthesespeciesto illuminate
preyand
communicate
withconspecifics
immunefromdetection
by potentialpredators
and preyalike (Partridge&
Douglas1995).
The thirdgenusof dragonfish
thatproducesfar-red
light
to long-wave
biolumi' Aqalacosteus
' is also sensitive
nescence,
although
themechanism
it employs
tobe so is
verydiffierent
fromthatusedby theothertwospecies.
Thus,although
Malacosteus
niger
possesses
twolong-waveshifted
visualpigments
(imaX
valuesca.520 and 540nm)
similartothosebasedon theshorter-wave
opsinonecan
extract
fromAristostomias
and Pachystomias
(figure
lb),it
lacksthesecondopsinthatenablestheotherspeciesto
havepigments
withimaX beyond550nm(Douglasetal.
1998b,1999). However,microspectrophotometry
has
shownthattheoutersegments
ofM.niger,
in addition
to
theirtwovisualpigments,
alsocontain
oneormoreadditionalpigments
withabsorption
maximaaround670nm
thatare notbleachedsignificantly
by light(figurelb;
Bowmaker
et al. 1988;Partridge
et al. 1989).We have
shownthatthesepigments
are,as suggested
byBowmaker
et al. (1988),used by M.nigeras a photosensitizer
to
enhanceitssensitivity
tolong-wave
radiation
(Douglaset
al. 1998b,
1999).Thus,wavelengths
aroundtheimaxofthe
photosensitizer
(671nm)are moreeffiective
at bleaching
Malacosteus
visualpigments
thanotherwavelengths
(i.e.
654nm)nearertheabsorption
maximum
of thevisual
pigments.
Therefore
lightcannotbe bleaching
thevisual
pigments
directly.
The photosensitizer
therefore
must
absorblightat itsabsorption
peakin thefar-red
and indirectly
bleachtheshorter-wave-sensitive
visualpigments.
Although
wedo notyetknowthemechanism
ofphotosensitization,
we havebeenableto identify
thepigments
involved
as a mixture
ofbacteriochlorophyll
derivatives.

P>lil.Wans.R. Soc.Lond.B (2000)

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Dietary
origin
ofretinalthotosensitizer
R. H. Douglasandothers 1271
especiallyin viewofthefactthatthevisualpigmentchromophores,and the astaxanthin-basedtapetumof this
species (Denton & Herring1971),are also derivedfrom
dietarysources.If theyhave access to chlorophyll,
the
two principal modificationsrequired to produce the
photosensitizing
pigment,hydrolysis
ofthefarnesyl
group
and demetallation,
can easilytakeplace in thealimentary
_,
!
tractsot organlsms.
The Malacosteus
photosensitizer
is, however,derived
frombacteriochlorophylls
c and d, whichare onlyknown
to occur in greenphotosynthetic
bacteria(orderRhodo'-/
\- spirillales,
suborder
Chlorobiineae,
families:ChlorobiaJ
<
ceae, bacteriochlorophylls
c and d; and Chloroflexaceae,
bacteriochlorophyll
c). Green bacteriahave been identified in subtidalmarine sedimentsbut not in the open
ocean. It is therefore
unclearhow theyare incorporated
intotheopen-oceanfood-chain
leadingto M. niger.
Nevertheless,M. niger,
has a mostunusualdietwhencompared
withitscloserelatives.
The stomiiddragonfish
are a relativelylargefamilyof deep-seafishesand most,including
Aristostomias
and Pachystomias,
eat mainly myctophids
(deep-sea lantern fish). In contrast, M. nigerfeeds
primarilyon euchaetidand aetideidcopepods (Sutton&
Hopkins1996),whichhavedirecttrophicaccessto photosynthetic
organisms.
We therefore
comparedthe fluorescent
excitationand
emissionspectra(recordedat room temperature
witha
Perkin-Elmer
LS50 spectrofluorimeter)
ofa suspensionof
(c)
retinalcells in 20% sucrose(figure2a), whichhighlights
the photosensitizing
pigment(Douglas et al. 1999),with
similarspectrapreparedfromhomogenates
and methanol
extractsof whole copepods (Euchaetasp.) (figure2b)
collectedin theGulfofMain and thestomachcontents
of
Malacosteus
caught in the same region (figure2c). The
spectraderivedfromthe retinalcell suspensionswere
typicalof a magnesium-free
chlorophyll
derivativeand
wereconsistent
withtheidentity
of thephotosensitizer
as
Chlorobium
pheophorbides(Douglas et al. 1999). The
spectraobtainedfromthecopepods (figure2b) werevery
350
750
450
550
650
similarto thoseof the retinalphotosensitizer
(figure2a)
I
wavelength
(nm)
consistent
witha commonorigin.Nevertheless,
copepods
are thoughtto consumeprimarilyphytoplankton,
which
Figure2. Fluorescentexcitation(dashedlines) and emission
containderivatives
of chlorophylls
a and b, not bacterio(solidlines) spectraof (a) an unpurified
M. nigerretinal
chlorophylls
c and d. Interestingly,
however,anaerobic
cell suspensionin 20% sucrosein PIPES-bufferedsaline
purplesulphurbacteriapossessingbacteriochlorophyll
(excitationat 418nm and emissionat 670nm) (afterDouglas
a
etal. 1999); (b) a methanolextractofwholecopepods
existin open-oceanecosystemsand have recentlybeen
(Euchaetasp.) (excitationat 400nm and emissionat 670nm);
describedin the gutsof some pelagic copepods (Proctor
(G) a methanolextractofMalasosteus stomachcontents)
1997).
(excitationat 420 nm and emissionat 670 nm).
The peak in the fluorescence
excitationspectrumat
670nm of the Malacosteus
stomachcontents(figure2c)
also suggeststhe presenceof a chlorophyllderivative.
Specificallythe photosensitizer
is composed of several However,the spectrumis more complexthan those of
Chlorobium
pheophorbides;that is a mixtureof defarne- pure chlorophyllderivativesor copepods due to the
sylatedand demetallatedderivativesof Chlorobium
chlor- presence of other, non-chlorophyll-related,
fluorescing
ophylls 660 (bacteriochlorophyll
G)
and Chlorobiumcompounds.Thus, whilewe cannotshowbeyonddoubt
chlorophylls
650 (bacteriochlorophyll
d), withthe latter that the origin of the Malacosteusphotosensitizer
is
predominating
(Douglas etal. 1998b,1999).
dietary,sincewe have not identified
the chlorophyll-like
One of the outstanding
questionsis the sourceof this substancesin the diet of Malacosteususing definitive
chlorophyll-derived
photosensitizer.Two alternatives methodssuch as mass spectrometry,
the fluorescence
presentthemselves:the animal either synthesizesthe spectraare highlysuggestive
ofit.
materialitselfor obtainsit fromthe diet.We knowof no
example of chlorophyll
derivativesbeing synthesizedin
The previously
published
workdescribed
wouldnothavebeen
vertebrates.A dietary origin thus seems more likely, possiblewithout
financial
aid fromtheNaturalEnvironment
(a)

/J

'

'

'

tt't

c)

c)
cK

st

Phil.Trans.
R. SOG.
Lond.B (2000)

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All use subject to JSTOR Terms and Conditions

ofretinalphotosensitizer
origin
1272 R. H. Douglasand others Dietary
Douglas, R. H., Partridge,J. c., Dulai, K., Hunt, D.,
Mullineaux,C. W., Tauber,A. & Hynninen,P. H. 1998b
393,423-424.
Aature
Dragonfishsee usingchlorophyll.
J.c., Dulai, K. S., Hunt, D. M.,
Douglas, R. H., Partridge,
Mullineaux,C. W. & Hynninen,P. H. 1999 Enhancedretinal
photosensiusinga chlorophyll-derived
longwavesensitivity
a deep-seadragonfishwithfarred
tiserin Malasosteus niger,
Res.39, 2817-2832.
Vision
bioluminescence.
In
to thedeep-seaenvironment.
Locket,N. A. 1977Adaptations
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inputfrom
ResearchCouncil and The Royal Society,scientific
D. M. Hunt,K. Dulai and P. H. Hynninenand supportfrom
and RRS Discovery.
the officersand crew of RRS Challenger
Thanks also to E. A. Widder,T. Frankand P. J. Herringfor
much-neededintellectualinput.The copepods and Malasosteus
stomachcontentsexaminedforthis studywere collectedwith
thehelpofRachel Ince,TraceySuttonandJoseTores.

Lond.B (2000)
R. SOG.
Phil.Trans.

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