Chlorophyll-Derivedretinalphotosensitizer of Malacosteus Niger

doi 10.1098/rstb.2000.0681

Long-wavesensitivityindeep-seastomiid dragonsh withfar-redbioluminescence: evidence for adietaryoriginof the chlorophyll-derivedretinalphotosensitizer of Malacosteus niger

R.H.Douglas 1*,C.W.Mullineaux 2 and J.C. Partridge 3 1Applied Vision Research Centre,Department of Optometry and Visual Science,CityUniversity ,311^321 Goswell Road, London EC1V7D D,UK 2Department of Biology,University College London, Darwin Building,Gower Street, London WC1E 6BT,UK 3School of Biological Sciences,University of Bristol,Woodland Road, Bristol BS81UG ,UK

Bothresidual downwelling sunlight and bioluminescence, which are the twomain sources ofillumination availablein the deepsea, have limited wavebandsconcentrated around 450^ 500nm. Consequently,the

wavelengthsof maximum absorption ( lmax)ofthe vastmajority of deep-sea ¢sh visualpigments also cluster inthis partof the spectrum. Threegenera of deep- sealoose-jawed dragon¢ sh ( Aristostomias , Pachystomias and Malacosteus ),however,inaddition to the bluebioluminescence typical of most deep-sea animals,also produce far-red light(maximum emission 4700nm) fromsu borbitalphotophores. All three generaare sensitive inthis partof the spectrum, towhich all other animals of the deepsea are blind,potentially a¡ ording them aprivatewaveband for illuminating prey and for interspeci¢ c commu- nicationthat is immune fromdetection bypredators and prey . Aristostomias and Pachystomias enhancetheir long-wavevisual sensitivity bythe possession ofat least three visualpigments that arelong-wave shifted (lmax values ca.515,550 and 590 nm) comparedwith those ofother deep-sea ¢shes. Malacosteus , on the otherhand, although it doespossess twoof these red-shifted pigments ( lmax values ca. 520and 540 nm), lacksthe most long-wave-sensitive pigments foundin the othertwo genera. However ,it further enhances its long-wavesensitivity witha chlorophyll-derivedphotosensitizer withinits outersegments. The£ uorescence emission andexcitation spectra ofthis pigmentare very similar tospectra obtainedfrom mesopelagiccopepods, which are an important component of diet of Malacosteus ,suggestinga dietary originfor this pigment. Keywords: ¢sh; visualpigment; bioluminescence ;photosensitizer; chlorophyll;deep sea

Thedeep sea is byfar the largesthabitat on Earth, shallowerdepths. Yet manyanimals, including most ¢shes, coveringover 60% of its surface andhaving an average livingwell below the reachof sunlight, have well- depthclose to 40 00m. Despite this, less is knownabout developedvisual systems. Thisallows them toview the it thanabout most other environments.However ,it has secondsource oflight in the deepsea: bioluminescence longattracted the attentionof vision scientists whohave producedby over 80% of the species inhabitingthis shownthat the eyes ofdeep- sea¢ shes displayan aston- region.Both residual sunlight and bioluminescence are ishingdiversity of anatomical adaptations to their unique spectrallyvery restricted withmost radiationbeing in the environment( Locket1977; W agner et al. 1998).Thedeep region450^ 500nm. Notsurprisingly ,therefore, the vast seahas also lured those withan interest invisual majorityof ¢ shes havevisual pigments withpeak absor- pigmentecology .One ofthe aims ofsuch workis to bance (lmax)inthis regionof the spectrum, providinga relate the spectral absorptioncharacteristics ofan goodmatch betweenenvironment and visual pigments, animal’svisualpigments toits spectral environment.The althoughdetailed analysis shows that evenhere the deepsea holds perhaps a specialattraction for such work relationshipis notstraightforward (Partridge et al. 1989; as,unlike in shallow water or on land, its photicenvir- Douglas et al. 1998a). onmentis inmany aspects quite simple. Thus,some Althoughthe wavelengthof maximumemission ofmost residualsunlight can penetrate the upper10 00mofthe deep-seabioluminescence occurs inthe blueregion of the oceanin ideal conditions, although in most waters spectrum, this is notalways so. The most strikingexcep- visiblesunlight is e¡ectively extinguished at signi¢ cantly tions arethree generaof deep-sea dragon¢sh ( Malacosteus , Aristostomias and Pachystomias ;orderStomiiformes, family *Author forcorrespondence (r.h.douglas@city .ac.uk). Stomiidae),which,in addition to blue bioluminescence

(a) Althoughsuch pigments area better match tothe far-red 1.0 bioluminescenceof these animalsthan the visualpigments ofmost deep-sea animalsthat have lmax valuesaround 0.8 480^490nm, the match betweenpigments absorbingopti- mallyat 51 5^550nm, andbioluminescent emissions peakingabove 700 nm, is still farfrom perfect. Usinga 0.6 retinalwhole-mount technique, however, which isolates pigments that donot survive retinal extraction or any form 0.4 ofpreservation(Douglas et al. 1995),wehave been able to e

c showthat both Aristostomias tittmanni (Partridge& Douglas n a

b 0.2 1995) and Pachystomias microdon (Douglas et al. 1998a) r o

s possess athird pigmentwith lmax around588^595 nm b a / (¢gure 1a).Thesepigments, whichare by some marginthe

n 0.0 o i most long-wave-sensitive rodpigments everdescribed, s s i (b) appear,basedon the shapeof their absorptionspectrum, m e 1.0 touse retinalas their chromophorebound to a second d e

z long-waveopsin. I twouldtherefore notbe unreasonableto i l a supposethat these retinas mightactually contain a fourth

m 0.8 r

o visualpigment composed of 3,4- dehydroretinalbound to n this long-waveopsin. Such a pigmentwould give a 0.6 virtuallyperfect match tothese animals’bioluminescence (¢gure 1a).Unfortunatelywe were unable to isolate such a 0.4 pigment,probably because tissue preparationswere made underdim red illumination,which would cause such long- 0.2 wave-sensitivepigments tobleach. Thus both Aristostomias and Pachystomias containthree, 0.0 andpossibly four ,verylong-waveshifted visualpigments 400 500 600 700 800 allowingthem tosee their ownbioluminescence, which wavelength (nm) wouldbe invisible to all other species. Sucha system potentiallyenables these species toilluminate prey and Figure 1. (a)Bioluminescenceof Aristostomiastittmanni (dashed communicatewith conspeci¢ cs immune fromdetection line;Widder et al. 1984),and the best-¢ t templates(solid bypotential predators and prey alike ( Partridge& lines)of thethree visual pigments so faridenti¢ ed in itsretina Douglas1 995). (arhodopsin ^porphyropsinpigment pair with lmax values 520 Thethird genusof dragon¢ sh that producesfar-red and551 nmanda rhodopsinwith a at588nm)(Partridge lmax light, Malacosteus ,is alsosensitive tolong-wave biolumi- &Douglas1995). The lighterline represents a theoretical nescence, althoughthe mechanism it employsto be so is porphyropsinwith lmax at669nm, which isthe `partner’ of thelong-wave-sens itiverhodopsin (calculated using a formula verydi¡ erent fromthat used bythe othertwo species. basedon empiricaldata relating lmax valuesof pigments in a Thus,although Malacosteus niger possesses twolong-wave- rhodopsin^porphyropsinpigment pair) (after Douglas et al. shifted visualpigments ( lmax values ca. 520and 540 nm) 1998a). (b)Bioluminescenceof M. niger (dashedline; Widder similar tothose basedon the shorter-waveopsin one can et al. 1984),and best-¢ t templates(solid lines) of thetwo extract from Aristostomias and Pachystomias (¢gure 1b), it identi¢ed visualpigments (a rhodopsinwith lmax 515 nm and lacksthe secondopsin that enablesthe otherspecies to aporphyropsinwith lmax 540nm).The absorptionspectrum havepigments with lmax beyond550 nm (Douglas et al. oftheputative photosensitize r(lighterline) is representedby 1998b,1999).However,microspectrophotometryhas apuri¢ed diethylether extract of aretinalsuspension (after shownthat the outersegments of M. niger,inaddition to Douglas et al. 1999). their twovisual pigments, alsocontain one or more addi- tionalpigments withabsorption maxima around 670 nm that arenot bleached signi¢ cantly by light (¢ gure 1 b; similar tothat ofotherdeep- seaanimals, also have subor- Bowmaker et al. 1988;Partridge et al. 1989).Wehave bitalphotophores producing far-red bioluminescencewith shownthat these pigments are,as suggestedby Bowmaker spectral emissions peakingsharply at wavelengths beyond et al. (1988),used by M. niger asa photosensitizer to 700nm(Denton et al. 1970,1 985;Widder et al. 1984). enhanceits sensitivity tolong-wave radiation (Douglas et We, andothers, haveshown, using conventional retinal al. 1998b,1999).Thus,wavelengths around the lmax of the extract spectrophotometry,that tofacilitate perception of photosensitizer (671nm) aremore e¡ective atbleaching their ownlong-wave bioluminescence, members ofall Malacosteus visualpigments thanother wavelengths (i.e. three generahave at least twovisual pigments that are 654nm) nearerthe absorptionmaximum of the visual long-waveshifted comparedwith those ofother deep-sea pigments. Thereforelight cannot be bleaching the visual animals (lmax ca. 515and550 nm) (e.g.Partridge et al. pigments directly.Thephotosensitizer therefore must 1989;Bowmaker et al. 1988;Partridge & Douglas1 995; absorblight at its absorptionpeak in the far-red andin- Douglas et al. 1998b,1999).Suchpigments forma directlybleach the shorter-wave-sensitive visualpigments. `rhodopsin^porphyropsinpigment pair’ based on the Althoughwe do not yet know the mechanism ofphoto- same opsin,which in some photoreceptorsis boundto sensitization,we have been able to identify the pigments retinaland in others to3,4- dehydroretinal. involvedas a mixture ofbacteriochlorophyll derivatives.

Phil.Trans.R. Soc.Lond. B (2000) Dietary origin of retinalphotosensitizer R. H.Douglasand others 1271

especiallyin view of the factthat the visualpigment chro- (a) mophores,and the astaxanthin-basedtapetum ofthis species (Denton &Herring 1971),arealso derived from dietarysources . If theyhave access tochlorophyll, the twoprincipal modi¢ cations required toproduce the photosensitizingpigment, hydrolysis of the farnesylgroup anddemetallation, can easily take place in the alimentary tracts oforganisms. The Malacosteus photosensitizer is, however,derived frombacteriochlorophylls c and d,whichare only known tooccur in green photosynthetic bacteria (order Rhodo- spirillales,suborder Chlorobiineae, families :Chlorobia- ceae,bacteriochlorophylls c and d;andChloro£ exaceae, bacteriochlorophyll c).Green bacteriahave been identi- (b) ¢edin subtidal marine sediments butnot in the open ocean.I tis therefore unclearhow they are incorporated intothe open-oceanfood-chain leading to M. niger. Never-

e theless, M. niger,hasa most unusualdiet whencompared c n

e withits closerelatives. The stomiid dragon¢sh area rela- c s

e tivelylarge family of deep-sea ¢shes andmost, including r o

u Aristostomias and Pachystomias ,eatmainly myctophids l f (deep-sealantern ¢ sh).Incontrast, M. niger feeds primarilyon euchaetid and aetideid copepods (Sutton & Hopkins1 996),whichhave direct trophicaccess tophoto- synthetic organisms. Wetherefore comparedthe £uorescent excitationand emission spectra (recorded atroom temperature witha (c) Perkin-Elmer LS50spectro£ uorimeter) ofasuspensionof retinalcells in20% sucrose (¢gure 2 a),whichhighlights the photosensitizingpigment ( Douglas et al. 1999), with similar spectra preparedfrom homogenates and methanol extracts ofwhole copepods ( Euchaeta sp.)(¢gure 2 b) collectedin the Gulf ofMainand the stomachcontents of Malacosteus caughtin the same region(¢ gure 2 c). The spectra derivedfrom the retinalcell suspensions were typicalof a magnesium-free chlorophyllderivative and wereconsistent withthe identityof the photosensitizer as Chlorobium pheophorbides(Douglas et al. 1999). The spectra obtainedfrom the copepods(¢ gure 2 b) were very 350 450 550 650 750 similar tothose ofthe retinalphotosensitizer (¢gure 2 a) wavelength (nm) consistent witha commonorigin. N evertheless, copepods Figure2. Fluorescent excitation (dashed lines) and emission arethought to consume primarily phytoplankton, which (solidlines) spectra of ( a)anunpuri¢ed M. niger retinal containderivatives of chlorophylls a and b,notbacterio- cellsuspension in 20%sucrose in PIPES-bu¡ered saline chlorophylls c and d.Interestingly,however,anaerobic (excitationat 418nmand emission at 670nm)(after Douglas purplesulphur bacteria possessing bacteriochlorophyll a et al. 1999); (b)amethanolextract of whole copepods exist inopen-ocean ecosystems andhave recently been (Euchaeta sp.)(excitation at 400nmand emission at 670nm); described inthe guts ofsome pelagiccopepods ( Proctor (c)amethanolextract of Malacosteus stomachcontents) 1997). (excitationat 420nmand emission at 670nm). Thepeak in the £uorescence excitationspectrum at 670 nm of the Malacosteus stomachcontents (¢gure 2 c) alsosuggests the presence ofa chlorophyllderivative. Speci¢cally the photosensitizer is composedof several However,the spectrum is more complexthan those of Chlorobium pheophorbides;that is amixture ofdefarne- purechlorophyll derivatives or copepods due to the sylatedand demetallated derivatives of Chlorobium chlor- presence ofother ,non-chlorophyll-related,£ uorescing ophylls660 (bacteriochlorophyll c ) and Chlorobium compounds.Thus, while we cannot show beyond doubt chlorophylls650 (bacteriochlorophyll d ),withthe latter that the originof the Malacosteus photosensitizer is predominating(Douglas et al. 1998b, 1999). dietary,since wehave not identi¢ ed the chlorophyll-like One ofthe outstandingquestions is the source ofthis substances inthe diet of Malacosteus usingde¢ nitive chlorophyll-derivedphotosensitizer .Twoalternatives methods such asmass spectrometry,the £uorescence present themselves: the animaleither synthesizes the spectra arehighly suggestive of it. materialitself orobtains it fromthe diet. Weknowof no exampleof chlorophyll derivatives being synthesized in The previouslypublished work described would not have been vertebrates. Adietaryorigin thus seems more likely, possiblewithout ¢ nancialaid from the Natural Environment

Phil. Trans.R. Soc.Lond. B (2000) 1272R. H. Douglasand others Dietary origin of retinalphotosensitizer

ResearchCouncil and The RoyalSociety ,scienti¢c inputfrom Douglas,R. H., Partridge, J .C., Dulai,K., Hunt,D., D.M. Hunt,K. Dulaiand P .H.Hynninenand support from Mullineaux,C. W.,Tauber,A. &Hynninen,P .H.1998 b theo¤ cers and crew of RRS Challenger and RRS Discovery. Dragon¢ shsee using chlorophyll. Nature 393, 423^424. Thanksalso to E. A. Widder,T .Frankand P .J.Herring for Douglas,R. H., Partridge, J. C., Dulai,K. S., Hunt,D. M., much-neededintellectual input. The copepodsand Malacosteus Mullineaux,C. W.&Hynninen,P .H.1999Enhanced retinal stomachcontents examined for this study were collected with longwavesensitivity using a chlorophyll-derivedphotosensi- thehelp of Rachel Ince, T raceySutton and Jose T ores. tiser in Malacosteusniger ,adeep-seadragon ¢ shwith farred bioluminescence .VisionRes. 39,2817^2832. REFERENCES Locket,N. A. 1977Adaptations to the deep- seaenvironment. In Handbookof sensory p hysiology ,vol.VII/ 5(ed.F .Crescitelli), Bowmaker,J. K., Dartnall, H. J.A. &Herring,P .J.1988 pp.67^192. Berlin: Springer. Longwave-sensitivevisual pigments in somedeep sea ¢ shes: Partridge,J .C. &Douglas,R. H.1995F ar-redsensitivity of segregationof `paired’ rhodopsins and porphyropsins. J. Comp. dragon ¢sh. Nature 375, 21^22. Physiol. A163, 685^698. Partridge,J .C., Shand,J., Archer, S. N.,L ythgoe,J. N. & Van Denton,E. J.& Herring,P .1971Reportto the council. J. Mar. Groningen-Luyben,W .A.H.M. 1989Interspeci¢ c variation Biol.Ass. UK 51, 1035. in thevisual pigments of deep- sea¢ shes. J.Comp.Physiol. Denton,E. J.,Gilpin-Brown,J .B. &Wright,P .G.1970 On the A164, 513^529. `¢lters’ in thephotophores of mesopelagic ¢ shand on a ¢sh Proctor,L. M. 1997Nitrogen-¢ xing, photosynthetic, anaerobic emittingred light and especially sensitive to red light. J. bacteriaassociated with pelagiccopepods. Aquat.Microb .Ecol. Physiol.Lond. 208, 72P^73P. 12, 105^113. Denton,E. J.,Herring, P .J.,Widder,E. A., Latz,M. F.&Case, Sutton,T .T.&Hopkins,T .L.1 996T rophicecology of the J.F.1985The rolesof ¢ ltersin thephotophores of oceanic stomiid( Pisces:Stomiidae) ¢ shassemblage of the eastern animalsand their relation to vision in theoceanic environ- Gulfof Mexico: strategies, selectivity and impact of a top ment. Proc.R. Soc.Lond. B 225, 63^97. mesopelagicpredator group. Mar. Biol. 127, 179^192. Douglas,R. H.,Partridge,J. C. &Hope,A. J.1 995Visual and Wagner,H.-J., F ro « hlich,E., Negishi,K. &Collin,S. P.1998 lenticularpigments in theeyes of demersaldeep- sea¢ shes. J. The eyesof deep-sea¢ shes.II. F unctionalmorphology of the Comp.Physiol. A177, 111^122. retina. Prog.Ret.Eye Res. 17, 637^685. Douglas,R. H.,Partridge, J .C. &Marshall,N. J. 1998 a The Widder,E. A.,Latz,M. I.,Herring, P .J.&Case,J. F .1984F ar visualsystems of deep- sea¢ sh.I. Optics, tapeta, visual and redbioluminescence from two deep- sea¢ shes. Science 225, lenticularpigmentation. Prog.Ret.Eye Res. 17, 597^636. 512^514.

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