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Reference: Bin/. Bull 181: 181-188. (August, 1991) Bioluminescence Maintenance in Juvenile Porichthys notatus ALLEN MENSINGER AND JAMES CASE F. F. Department ofBiological Sciences andMarineScience Institute, University ofCalifornia, Santa Barbara, California 93106 m Abstract. Bioluminescence in the midshipman fish. waters to 400 on the coastal shelf except during the Porichthysnotatusfrom theSanta Barbaracoastal region, breeding season. Then, during late spring and early sum- was quantified from onset through the first two years of mer, sexually mature adults migrate inshore to spawn in life. Maximum light emission was 2.5 X 109 photons s~' the intertidal to 80 m depths (Feder et al. 1974). Males upon leavingthe nest and reached 2.0 X 10' photonss~" establish sheltered nesting sites and acoustically attract within the first year. These intensities may be sufficient females. Females deposit up to 400 eggs, and multiple for counterillumination in moon or starlight over most matings may result in the maleguarding nestscontaining of the depth range of the fish. The bioluminescence of more than 1000 eggs. juvenilesrecently detached from the nest wasdepleted by P. notatus ranges from southeastern Alaskan waters to multiple topical applications ofa dilute noradrenalin so- Baja California (Wilimovski, 1954) with a discontinuity lution. A luciferin-free diet also exhausted luminescence alongthecoastofOregon dividingthegroupintonorthern in 10-18 months. Bioluminescence was restored within and southern divisions (Warner and Case, 1980). Al- 24 h after feeding depleted fish with dried specimens of though the photophores ofboth groups are ultrastructur- thebioluminescent marineostracod I'argulahilgendorfti. ally indistinguishable, the northern fish are non-lumines- and light emission capacity was correlated with the cent (Strum, 1969). Both populations contain luciferase, amount consumed. Predation by second year fish (18 but the northern population lacks luciferin (Tsuji et al., months) uponjuvenile P. notatus(3 months)orupon live 1972; Barnes et al.. 1973). Administration ofthe biolu- I". tsujiialso restored luminescence. Afterrestoration, luminescent marine ostracod I'argula hilgendorfti, induces minescencegraduallydisappearedwithinseveral months. luminescence in Puget Sound fish (Tsuji et al.. 1972; Consumption of luciferin-containing organisms by al- Barnes et al.. 1973). ready competent fish did not increase light intensity. Ju- Fish ofthe southern division, south ofMonterey Bay, venile P. notatus from the Santa Barbara coastal region are uniformly luminescent, while the central population, require exogenous luciferin to remain luminescent. north of Monterey to Cape Mendocino, includes luminescent and non-luminescent fish (Warner and Case, Introduction 1980; Thompson and Tsuji, 1989). The analog of V. hil- The midshipman fish, Porichthysnotatus. hasbeen the gtseunjdiio,rfiwihoasleongditshteriwbeutsitoenrncoNionrctihdeAsmewirtihcatnhecosaosuttihserIn'. subject of many investigations since Greene ( 1899) first midshipman population (Kornickerand Baker, 1977). Its dneotremdalitsphboitoolpuhmoirneess,cepnrciemaorriilgyincaotnifnginferdomtohtuhnedrveednstraolf atsbusjeiincisetfhreolmucniofretrihnersnouwracteerfsorletdhetososuptehceurlnatipoonputlhaattioIn'. surface. Its natural history has been reviewed by Hubbs (Warner and Case, 1980). (1920), Arora (1948) and Ibara(1967). A nocturnal pred- The inability to experimentally deplete luminescence ator, it remains buried in the sand during the day and in luminous adults despite long periods ofcaptivity and ascends at night to feed. The habitat is moderately deep repeatedchallengeswith noradrenalin suggested that adult P. notatus have already acquired large luciferin reserves Received 17 December 1990;accepted21 March 1991. from the diet or that there is a luciferin recycling or syn- 181 182 A. F. MENSINGER AND J. F. CASE thesismechanism (Barnesela/., 1973). Ingestion ofsmall 1W.V numbers of luminescent ostracods rendered northern adults capable ofluminescence for up to two years, with light yield greater than was considered theoretically possible for the amount of luciferin consumed, suggesting that de novo synthesis is triggered by exogenous luciferin orthat there isa recycling mechanism (Thompson el al, 1988a). A preferential mechanism for rapid uptake ofluciferin from the digestive system has also been reported in P. notatus (Thompson el al., 1988b). The purpose ofour study was to observe the effects of a luciferin-free diet on the development of bioluminescence in P. notatusjuveniles ofthe southern population and to ascertain whether the fish ofthis population need exogenoussourcesofluciferin toremain luminescent. To this end, light emission was quantified from its onset in larval fish through the first two years in laboratory-reared and in locally collected individuals ofP. notatus. The effects ofmultiple challenges with noradrenalin on the depletion of luminescence in the fish are described as are experiments that contributed to the evaluation ofthe luciferin recycling hypothesis. Materials, Methods and Results Collection Breeding pairs ofP. notatus were collected intertidally along the Pacific Coast,just north ofSanta Barbara, Cal- ifornia. They were maintained in large aquaria with sand filtered, runningseawater(16-20C). Masonryand large, inverted abalone shells provided nesting sites. Mating usually ensued duringthe first night in captivity and produced between 100-400 eggs. Femaleswere removed after spawning and males were retained to maintain the nests. Additional nests with guardian males were obtained by divers. After larval detachment (35-50 days post-fertil- ization),juveniles were placed in aquaria with sand-cov- ered bottoms sufficient for burrowing. Free-livingjuveniles were collected with a twenty-five foot, semi-balloon ottertrawl from Octoberto May. Min- imum depth ofcapture offirst year fish ranged from 30 m m in the fall to 80 in late spring. All fish were maintained on a luciferin-free diet consistingof\\v\ngArteniia and kelp mysids and frozen squid. Laboratory reared and trawled fish of the same year class had similar growth rates (Fig. 1A). Photophore diameter increased directly with standard length (Fig. IB). Light meaxnrenicni Bioluminescence was quantified with an integrating sphere quantum counting photometer (Latz et al.. 1987). Small fish, lessthan 4 cm si (standard length) wereplaced individually in a head down position in a clearplastictest BIOLUMINESCENCE IN P. NOTATVS 183 1E11 mately 40 min later. Exposure to fresh noradrenalin so- V lutionsduringthe trial did not appreciably raise intensity levels. 1 A 365 nm emitting ultraviolet (UV) lamp (UVSL 25, *r Ultraviolet Products Inc.) was used to qualitatively test o forluciferin in photophoresby observingfluorescence vi- o sually (Barnes et a/.. 1973). 1E09 Bioluminescence depletion and induction Luminescence was initiallydetectedabout 30daysafter 1E08 spawning in 1.7 cm si larval fish still attached to the nest. Bioluminescence capacity in the laboratory population maintained on a luciferin-freediet increased for6 months, before gradually declining (Fig. 2). Loss ofluminescence III!, capacity occurred from 10 to 18 months after spawning. 100 200 300 400 500 600 Laboratory reared and trawled fish showed indistinguish- TIME (DAYS) able luminescence levels up to approximately 300 days. Figure 2. Bioluminescence induction and depletion. Maximum in- Recently detached juvenile fish (2.5-3.5 cm si) were tensity(photonss~')versustime(days)afterfertilization. Datarepresent exhaustedMofluminescence capacity by immersing them laboratoryraised(circles)andcaptured(squares)fish. Laboratoryraised in 0.005 noradrenalin for approximately 5 min every tfiwsohrweeceekisveadftneorceaxpotgueren.ouFsorlueciafcehrignrionupthteesdtieedt.,Tmreaawnlevdalfuieshswaerereshteoswtne;d otherday until no luminescent response waselicited(Fig. errorbarsrepresentoneSDofmean. Samplesizeranged from 10to25. 3). Depleted animals remained nonluminescent unless Dottedlinerepresentsnoisebackgroundofintegratingsphereandpoints their diet was supplemented with luciferin as described on line are tobeconsidered nonluminescent. below. The remaining untreated population was tested bimonthly forluminescence with noradrenalin. Foreach sphere. Larger fish were placed in a 7.0 cm diameter cir- sample ofthe latter group, the tests represented the first cular plexMiglass chamber, 3.5 cm high, containing 40 ml exposure to noradrenalin. of0.005 noradrenalin. Noradrenalin-depleted fish invariably could be restored Bioluminescence was recorded for 5 min with a RCA to luminescence competence by the administration of 8850 photomultipliertubeoperated at -1700 V and baf- fled so that only light reflected from the sphere internal surface was measured. This procedure is essential to ac- 1E-HOp- curately measure total emission from large organisms o containing many non-isotrophically radiating photo- cLnU cphroirmeisn.atTohrecadleitbercattoerdsaitgn-al0.w3a1s5pVroacensdsetdhethrresouulgthinag dfirse-- Oo 1E+09 CONTROL quencysignalwascountedwithanOrtecNo. 776counter/ , SVN PN 1 timeranddisplayedonaNorlandNo. 5400multichannel \ ;"*-* PN-2--- analyzer(MCA). Radiometric calibration ofthe detection %o '' PN3 ', system involved determination ofthe combined spectral responsitivity ofthe integrating sphere and photomulti- plier tube using an Optronic Laboratory Model 310 calibration source. Lightabsorbance byfish within thesphere wtoasbechneecgkliegdibalgea.inTshteaCM1C4Aacttirvaacteedwpahsossptohroerdeoanndfiflomunodr 10TIME (DAYS) 20 30 floppy disk. Figure 3. Noradrenalin induced depletion of bioluminescence. Immersion in noradrenalin solution elicited biolumi- Maximum intensity(photonss ')versustime(days)afterfirstchallenge nescence within 20 s by transcutaneous absorption as with noradrenalin. Forcontrol grouptested (squares), mean valuesare shown by the effectiveness oftopical application in air, shown;errorbarsrepresentoneSDotmean. Datapointsrepresenttime thus avoiding mouth and gills. Luminescence was not offirstchallengewithnoradrenalin.Control fishreceivedonlyonechal- obsMearvxeidmpurmiorlingohrtaedmriesnsailoinn wapapsliucsautailolny. attained within lrlieennpegreer.seepFnroterssetanhtceshatclhalrleecneugleea.xtepSdearlmiempaelsnte-tsasqliuzaefrieosshfl(ecoaigcrahcrliectsohnamtnircdolrterggirranoegsulspeisow)n,aosefa1ic0nh.tepSnoosilinittdy 5 min and gradually declined until exhaustion approxi- on timeaccordingtoequation y = 2.3 X 109 + 0.02x, r- = 0.61. 184 A. F. MENSINGER AND J. F. CASE dried Vargula hilgendorfii (0.7 to 4.0 mg). Depleted fish VAHLIGVUELA PJOURVIECNHITLHEYSS FREVEAZHEGDURLIAED CONTROL wereanesthetizedin tricaine methanesulfonate(MS222, ICN-K&K Laboratories), 100 mg/1 filtered seawater, and fed6-12whole,dried I'argula hilgendorfiiviaintramedic tubing (PE 190) slipped into the anterior gut. Controls were anesthetized but not fed. To guard against regurgi- tation, fish were monitored for 15 min after regaining their ability to swim. The few ostracods expelled were dried and their weight subtracted from the total. Restored luminescence neared but neverexceededpre- vious maximum output. Restoration was evident within 24 h, but maximal light output was usually not attained for several weeks. Total light emission was related to the amount of ]'argitla administered (Fig. 4). Naturally depleted, second year P. notatus (9.0 cm si) also had luminescence capacity restored by ingestion of dried Vargitla hilgendorfii (5.0-9.0 mg), live I', tsujii (n = 3) and recently detached juvenile P. notatus (n = 3 to 5) (Fig. 5). Forthe live feedings, fish were placed in small aquaria containing either three live Jr. tsujii. or five recently detachedjuvenile P. notatus (2.5 cm si) and monitored until prey were consumed. Restored luminescence neared but never exceeded previous maximum output. Living I'argulaproducedthe most rapidonsetandgreatest intensities. Reinduction was not permanent, and luminescence capacity gradually disappeared with time (Fig. 5). The rise indicated afterday 90 in Figure 5 forjuvenile P. notatus feeding on other P. notatus is an aberration representing one fish that had been gradually losing its capacity for luminescence. The other three fish were depleted, but died before testing on day 120. Ingestion of live I'. tsujiibyalready competentjuvenilesdid not result in increased light output. OI 2E-I-08 ffi+O~g MAXIMUM BIOLUMINESCENCE IN P. NOTATUS 185 o o O 11/85 =J 1E+09 186 A. F. MENSINGER AND J. F. CASE illumination. Although the P. notatusemission spectrum Although it has long been speculated that I'. tsujii is (Tsuji el al.. 1975) does not exactly match nocturnal as- thesourceofP. notatusluminescence, thisisthefirststudy tronomic light (Munz and MacFarland, 1977). this may in which fish were found to actively prey on (". tsujii, be unimportant because many oftheCalifornia nearshore albeit in laboratorytanks. Cannibalism wasalsoobserved fishesthat feed primarilyatnight lackthe visual pigments forthe firsttime in P. notatus. First yearfish areeasy prey necessary to detect hue differences (Hobson el al.. 1981). ofolder fish, and small conspecifics have been noted in Additionally, the variability in waterquality ofnearshore the stomach of the closely related species, P. myriaster waters can alter the apparent emission spectrum. Lumi- (Allen, 1982). Previously it had been thought that young nescence intensity is sufficient to counterilluminate moon fish mustaccumulateadequatelife-timeluciferin reserves or starlight throughout most ofthe normal depth range. before reaching maturity (Warner and Case, 1980), be- Upon leaving the nest, fish emit at 2.0 X 104 photons s~' cause it seemed unlikely that larger fish would consume cirT:, which is sufficient to counterilluminate starlight at small ostracods and would, at any rate, occupy deeper m m approximately 30 depth and full moonlight at 70 water outside the range of 1'argula. Clearly, such is not depth in Class 1A oceanic water (Jerlov, 1976) on cloud- the case and the overlapping ranges of first and second less nights. The fish probably could match intensities at year fish offSanta Barbara make it possible for younger somewhat shallower depths because the nests are found fish to be an additional exogenous source ofluciferin for in water usually less clear than class IA and the effects of older conspecifics. thus establishing a link which would kelp canopies or clouds are considerable. allow the larger fish effective indirect access to I'argula Although counterillumination in fish has not been luciferin. demonstrated in captivity, its presumed importance may It was calculated using the methods of Thompson et explain the early onset ofluminescence in larval fish that al.. ( 1987) that the fish should theoretically yield approx- become competent to luminesce weeks before detach- imately 2 X 10'5 photons s~' for the amount of dried ment. Given the inverted position ofthe larvae, low re- I'argiila ingested. Although light emission was only flectivity ofthe substrate and presence ofaguardian male, quantified for 5 min, none of the induced fish emitted it isdoubtful that luminescence has much functional sig- light for over 40 min and many displays lasted less than nificanceonthenest. However, it maybevitaltocounter- 20 min towards the end ofthe study. Even using the ex- illuminate immediately after detachment. tremely liberal calculation ofmultiplying the maximum This study shows, for the first time, depletion of lu- intensityattainedduringtherunby40 mintoderivepho- minescence in previously competent fish. Other investi- ton yield, and then summing the trials for each fish, the gatorshadbeen unabletodeplete naturallyluminousadult brightest fish only produced 2 X 1013 photons s ' (range fish orostracod-induced northern fishdespite longperiods 2 X 10i: to 2 X 10" photons s '), a value two orders of of captivity or multiple challenges with noradrenalin magnitude below the theoretical yield. Thompson et al. (Tsuji et til.. 1972; Barnes ct al.. 1973; Thompson et al., ( 1988) report that the fish retain approximately 1% ofthe 1987). In our study, luminescence was depleted by fre- luciferin ingested. Even after estimating the luciferin re- quent challenges with topically applied noradrenalin or tention rate at two orders ofmagnitude less than actually by maintenance on a luciferin-free diet. Noradrenalin ingested, the majority ofthe induced fish fall short ofat- treatment depleted luminescence within three weeks in taining theoretical values. recentlydetached fish, thusleadingtotheconclusionsthat These experiments do not support either a luciferin frequentstimulationexpendsmaternallyacquired reserves synthesis or recycling mechanism for juveniles of the and that a regeneration process isnot present. Injuvenile southern population. Luminescencecouldbedepleted by southern fish, lack of exogenous luciferin also depleted continual challenges with noradrenalin or maintenance luciferin reserves within 10-18 months. The depletion on a luciferin-free diet. Although luminescence could be may have been the result of spontaneous luminescence restored with exogenous sources of luciferin, the effect ormetabolicelimination ofluciferin. Lossofluminescent was not prolonged and emission values fell short oftheo- capacity by whatever means was not detrimental to the retical values. photophores as luminescence could be restored by lucif- These resultscontrast with the reportsofThompson et erin administration. al. (1987; 1988a,b) who found that the persistence of T. The three luciferin sources used in ourexperiments all hilgenclorfii [l4C]luciferin in the photophores and long induced luminescence between 20 and 48 h, more than lasting luminescence after feeding small amounts of lu- twice as quickly as reported in previous studies (Tsuji et ciferin suggested an active recycling mechanism in Puget al.. 1972; Barnes ct al.. 1973). This is attributed to the Sound adults (Thompson et al.. 1988b). Furthermore, smaller body size ofthe fish, which presumably allowed they reported I'argula feeding to previously non-lumi- quicker substrate transport to the photophores, and im- nescent fish, yielded more light than was theoretically proved instrumentation. possible for amount ofluciferin ingested. B1OLUMINESCENCE IN P. NOTATVS 187 Amajordifferencebetweenourinvestigationsandthose pub.). Whetherthisisdueto smallerreserves in posterior ofThompson el al. (1987; 1988a,b)wasour use ofsouth- photophores ora transport mechanism that gives the an- ernjuvenilesinsteadofnorthernadults. Thesepopulations terior sites higher priority remains unknown. A mecha- may have evolved different luciferin pathways in response nism favoringsupply ofluciferin to anterior ventral pho- to availability ofexogenous luciferin in the diet ofadults. tophoresduring luciferin limitation would have adaptive The southern habitat includes two luciferin sources, V. valuein preservingcounterillumination capability forthe tsujii and young P. notatus. Perhaps owing to this, they larger and therefore more conspicuous, anterior regions. havelostorhaveneverevolvedan alternative mechanism, Ifthe latter is true, however, the localized anterior nor- orpossibly itdoesnot function untiltheirlater, deepwater adrenalin injection sites used in Thompson et al. (1987) stage when they are out ofrange ofexogenous sources. If may have disproportionately depleted luciferin reserves \'argula disappeared gradually from the northern range, from the anterior series, thus resultingin the transport of the northern fish might have evolved mechanisms to luciferin from the more distal sites. Whatever the mech- maximize the effects ofincreasingly rare encounters with anism,extrapolatingintensitiesfromanteriorphotophores luciferin sources. Anotherpossibility isthat the pathways would, therefore, result in erroneously higher emission require minimal amounts ofluciferin to function. Once estimates and thereby induce error into calculations of levelsdropbelowacritical concentration, enzymesneeded luciferin use. for synthesis or recycling are no longer produced. We found no evidence ofa mechanism for long-term A second difference in these investigations is that the maintenance ofluminescence capacity in southern fish. use of the integrating sphere photometer allowed us to Eventuallossofluminescenceinanimalswithoutprevious quantify more accurately the total light emission per fish noradrenalin exposure rules out the possible deleterious andavoidtheassumptionsconcerningtotal lightemitted, effects ofrepeated noradrenalin challenges in the fish used average intensity, and differential photophore emission in our investigation. The loss of luminescence capacity made in previous reports (Thompson el al., 1987). For after induction with luciferin showed that there was no example, Thompson el al. (1987) multiplied the directly synthesis mechanism dependent on priming with non- measured intensity of 127 photophores mostly from the maternal sources of luciferin. The loss of maternally or branchiostegal and gular series (Greene, 1899) by 4.3 to naturally acquired reserves through spontaneous lumi- determine light emission from the whole fish, with the nescence, diffusion, or autoxidation may have decreased underlying assumption that all photophores emit equal luciferin below recyclable levels. However, the relatively intensities. However, we find that the branchiostegal and short reinduction periods (3-6 months) and low photon gular series contain the most intensely emitting photo- yieldscastdoubtonthepresenceofarecyclingmechanism phoresoftheentire fish (Table I). Wecalculated emission in second year southern fish. based on photophore average intensity by dividing the Weconcludethat fish ofthesouthern population must fish into three areas: ventral photophores inside the light continually acquire exogenoussourcesofluciferin, at least capture geometry ofthe Thompson et al. (1987) photo- during their early life history, to remain luminescent. multiplier (branchiostegal and gular series; n = 127), remaining ventral and lateral photophores (mandibular, Acknowledgments = pvehnottroalp,hoarnedsl(astecraaplulsaerriesse;rines; n39=7)1a50n)d. hWeeaddaentderdmoirnseadl J. GJ..WMaorrnienraconndtrAi.buHtuevdatrodthkeinedalrylypprhoavsiedsedofltihviisngstVuadyr.- total light output by taking the average intensity ofthe gii/u. J. G. Morin also most helpfully reviewed the manu- photophorescontainedwithin representativeseries(listed script and provided other useful comments. A. Uriu and above for each area) and multiplying the average photo- L. Trebasky helped with animal maintenance. Research phore intensity by the approximate number of photo- was supported in part by Office ofNaval Research Con- phores contained within each area. Our values indicate tracts N00014-84-K-0314 and N00014-89-J-1736. that quantification oflight intensity forthe whole animal by extrapolating from only the branchiostegal and gular Literature Cited photophores overestimates light emission by at least a Allen, M. J. 1982. Functional structure ofsoft-bottom communities factor of3.5. ofthesouthernCaliforniashelf.Ph.D.thesis.UniversityofCalifornia, Another factor not considered by Thompson et al.. San Diego, LaJolla,California. (1987) in arguing for luciferin recycling is that, in the Anctil, M. 1977. Development ofbioluminescence and photophores courseoflosingluminescencecapability, fish initiallylose in the Midshipman fish, Porichlhvs notatus. J Morptwl. 151: 363- luminescencecapacityinposteriorphotophores. Fishlow 396. in luminescence capacitywere notedby dark adaptedob- Arora, H.L. 1948. Observationsonthehabitsandearlylifehistoryof thebatrachoid fish. Porn-hthys notatusGirard. 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