Helgol Mar Res (2007) 61:55–66

DOI 10.1007/s10152-006-0053-4

ORIGINAL ARTICLE

The spatial pattern of bioluminescent Xashes in the polychaete Eusyllis blomstrandi (Annelida)

S. A. Zörner · A. Fischer

Received: 21 August 2006 / Revised: 23 November 2006 / Accepted: 23 November 2006 / Published online: 22 December 2006© Springer-Verlag and AWI 2006

Abstract Each of the trunk segments of the poly-chaete Eusyllis blomstrandi is equipped with pairedepidermal luminescent domains. They luminesce uponmechanical or electrical stimulation. Light emissioncan be rapidly turned on and oV, appears intracellularand is highly coordinated among the trunk segments.Luminescent light is typically emitted in series of Xas-hes. Light emission in a Xash starts locally in a group ofsegments and recruits adjacent segments at a rate asfast as ·1 ms/segment. The collapse of light emission atthe end of a Xash is almost simultaneous in all of thesegments involved. In the intact worm, the luminescentreaction usually involves only a posterior group of seg-ments. Facilitation becomes manifest as the consecu-tive Xashes in a series increase in brightness andduration and recruit additional anterior segments thatwere not active in earlier Xashes. The Xash series stopsabruptly instead of decreasing asymptotically in bright-ness. In posterior fragments, all the segments partici-pate in Xashing luminescence, indicating the loss of aninhibitory eVect exerted by the anterior end in the caseof whole animals. Posterior fragments survive and arestill capable of luminescence weeks after fragmenta-tion although they do not regenerate a head. Immedi-ately upon fragmentation of the worm, the posteriorfragment luminesces continuously for some secondswhile the anterior part quickly stops light emission.

This suggests a decoy and/or a predator-alerting func-tion of prolonged, strong luminescence by the mori-bund posterior fragment to the beneWt of the survivalof the anterior fragment.

Keywords Bioluminescence · Eusyllis (Polychaeta) · Segmental light organs · Facilitation · Predator alert

Introduction

Many species of polychaetes display bioluminescence.Best-known are bioluminescent phenomena in scaleworms (Aphroditoidea), in the parchment worms(Chaetopteridae), and in the syllids (Syllidae), some ofwhich are known as “glowworms” (e.g. Odontosyllisluminosa, Gaston and Hall 2000) or “Wreworms”(Odontosyllis enopla; Fischer and Fischer 1996). For anumber of other polychaetes, like the holopelagicgenus Tomopteris, bioluminescence has only beennoted (Dales 1971).

Spontaneous light emission in polychaetes has onlybeen reported from mature Odontosyllis species(Fischer and Fischer 1996; Gaston and Hall 2000) dur-ing their swarming excursions. Field observations oftheir sexual activity leaves little doubt that light emis-sion by the Odontosyllis female enables the male tolocate and meet its sexual partner. In other polychaetesas well as in immature Odontosyllis (Fischer andFischer 1996), bioluminescent activity seems to occuras a response to mechanical irritation, like touching theworm or mechanically shocking the substrate, and, inChaetopterus (Martin and Anctil 1984), to electricalstimulation of the ventral nerve cord or of isolatedparapodia. Light emission in these cases is interpreted

Communicated by H.-D. Franke.

S. A. Zörner · A. Fischer (&)Zoological Institute, University of Mainz, 55099 Mainz, Germanye-mail: aWscher@uni-mainz.de

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as an alarm response serving a deterrent or a decoyfunction (Morin 1983).

A variety of kinetics is observed in polychaete lightemission. A luminescent glow (>2 s duration; Hastingsand Morin 1991) emitted from secretions is found inChaetopterus and in swarming female O. enopla(Fischer and Fischer 1996) and O. luminosa (Gastonand Hall 2000). Emission of serial light Xashes, on theother hand, is ascribed to intracellular light productionand has been long-known in the scales of scale worms,in swarming male and immature Odontosyllis (Fischerand Fischer 1996), and in other luminescent syllidslike Pionosyllis pulligera (Bassot 1979) and Eusyllisblomstrandi.

Research on polychaete bioluminescence has so farfocussed on behaviour and biorhythmicity in polynoidscale worms, Chaetopterus, and Odontosyllis (Sylli-dae), and, in Chaetopterus, on the role of the nervoussystem in eliciting light production. Among the poly-chaetes, detailed studies on histology and Wne structureof light-emitting cells and organelles and on the tempo-ral and spatial patterns of light emission only exist forthe isolated scales of polynoids. These “scales” are spe-cialized parapodial appendages which are readily shedby autotomy and are then easily handled in the labora-tory. A considerable body of knowledge on structuralconditions and physiological parameters has accumu-lated for this experimental material (Pavans De Cecc-atty et al. 1977; Bilbaut and Bassot 1977; Bassot andBilbaut 1977a, b; Bassot 1987).

In intact polychaetes, the temporal and spatial pat-terns of bioluminescent Xashing, however, are still notprecisely known. This is due to the technical problemsposed by the low intensity of the luminescent light sig-nals and by the rapidity of the changes in their intensityand spatial pattern. The only exception is a brief reporton serial light Xashes emitted by the syllid P. pulligeraupon electrical stimulation (Bassot 1979). Recent tech-nical progress now allows high-speed video imaging atextremely low light intensities, and modern data man-agement enables easy processing of the raw materialfrom video- and photomultiplier recording.

We have carried out the study of bioluminescence ofthe syllid polychaete E. blomstrandi, a species closelyrelated to the genera Odontosyllis and Pionosyllisamong the Eusyllinae. E. blomstrandi can easily be col-lected around the island of Helgoland in the North Sea.As in other Eusyllinae, its light is emitted from pairedsegmental spots. We have studied the spatial and tem-poral patterns of bioluminescence by recording theluminescent light and show that these patterns cannotbe resolved by the unaided human eye. Here, we char-acterize the kinetics of bioluminescence in Eusyllis

under several diVerent conditions. The Wlms fromwhich most of the present images were taken are acces-sible on-line (Zörner and Fischer 2006).

Materials and methods

The animals

The polychaete E. blomstrandi occurs on the coasts ofthe Northern Atlantic. A dense population is found inthe phytal on the coast of the island of Helgoland in theNorth Sea where the worms construct and inhabit silktubes on the thalli of the red alga Delesseria sanguinea.Such thalli were collected at about 7 m depth by diversof the Biologische Anstalt Helgoland/AWI Bremerha-ven and placed in containers while underwater. In thelab, the algal thalli were transferred into shallowdishes, covered with a new change of seawater and leftwithout aeration. The worms then left the thalli, someimmediately after transfer, and some during the fol-lowing 24 h. They were then collected with a glasspipette and transferred into glass bowls or transparentplastic containers. Other polychaetes and nemerteanswere sorted out under a dissecting microscope to keepthem from attacking the Eusyllis.

Eusyllis blomstrandi can be transported and kept formonths in transparent round Xat-bottom plastic contain-ers with a lid. To avoid intraspeciWc attacks and canni-balism, worms must not be kept too crowded: e.g. threeworms in a 100-ml container Wlled with 15 ml seawaterwill do well. For transport, the worms are placed in suchcontainers at least 1 day prior in order to permit tubeconstruction overnight, providing them with a retreatduring the buVetings of transport. E. blomstrandi mustnot be exposed to temperatures higher than 18°C exceptfor brief periods of handling and experimentation. Sun-light or microscope illumination may quickly warm thecontainer temperature beyond the upper threshold oftolerance. For maintaining E. blomstrandi in the lab forweeks or months, a dense suspension of living brineshrimp (Artemia) larvae can serve as food.

Video imaging: handling the worms

A single Eusyllis worm is placed in the centre of a circu-lar cover slip 42 mm in diameter. Some drops of seawa-ter are added to form a small pond (about 20 mm indiameter) in the centre of the cover slip. The cover slip isplaced in a 100-mm plastic Petri dish on two plastic barsupports, and the Petri dish is stored in a wet chamber (a20 cm £ 20 cm £ 6 cm refrigerator plastic container witha seawater-wetted Wlter paper on the bottom). The

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worms are then left overnight in the dark where most ofthem will spin tubes for dwelling. In preparation forvideo imaging, such preparations are brought into adarkroom for at least 20 min and for the following arehandled under dim red light or in the dark.

The cover slip with a worm in its tube is placed in acell culture chamber (POC chamber system; H. Saur,Reutlingen, Germany). Two silicone gaskets (42 mm indiameter, 2 mm high) are placed on the cover slip andpressed against it by a stainless steel ring screw. Theresulting trough (3.6 ml volume) is Wlled with seawaterand left without cover. In the dim red light from a Wbreoptic illuminator with a red Wlter, the whole device isbrought into position for video imaging on a rotarymicroscope stage. The same red light source is used toilluminate a white diVuse light reXector mounted belowthe microscope stage. This arrangement provides theminimum background illumination that is needed toidentify the contours of the worm in the video Wlms.

Light emission is stimulated in the dark either in adiVuse manner by shocking the cover slip with a metalrod or as a localized stimulus by touching the worm witha needle. Alternatively, luminescent light emission canbe elicited by placing a worm between two Weld elec-trodes that are operated using square wave pulses at adirect voltage of ¸5 V. Autotomy is induced by pressureexerted with the curved cutting edge of a scalpel.

Video imaging: optics and cameras

The macro lens systems LUMINAR 40 (f = 40 mm)and LUMINAR 16 (f = 16 mm) (ZEISS) were used forvideo imaging in combination with a variety of exten-sion tubes. For this study we used the HAMAMATSUvideo camera system C9100-02, a particularly light-sen-sitive camera with a maximum spectral sensitivitybetween wavelengths of 500 nm and about 700 nm.

At its full resolving power of 512 £ 512 pixels, thiscamera yields 35.8 complete frames/s. However, when abinning rate of 4 £ 4 pixels is applied, the camera chip isread more quickly such that as many as 118.2 frames areread per second, though at the expense of image resolu-tion. The eVective “shutter” interval is inXuenced by thestorage process and is subject to some variation accord-ing to the nature of the image motifs. In our material,this interval varied between 8.46 and 12 ms and, in total,came very close to an average of 10 ms so that a “10-ms”interval was selected as the approximate “shutter” fre-quency in our video image series (Figs. 4, 8) and dia-grams (Figs. 5, 6, 7, 9, 10); the actual time intervals took,however, 9.71 ms on average.

For each one of the recorded pixels of every videoimage, a numerical value on the half-tone scale of up to

14-bits is stored. For use in the graphs inFigs. 5, 6, 7, 9, and 10, the amount of locally emittedlight was estimated from the video images and manu-ally symbolized by three levels of shading: dim (heavyshading), medium, and full intensity (no shading).

Video imaging: calibrating the camera and the background illumination

In order to adjust the video camera to the intensity ofthe light Xashes emitted by Eusyllis, some Xashes ofluminescence were recorded by a photomultiplier tube(PMT), type IP28 (BURLE Industries, Lancaster, PA,USA). Using the PMT, an LED light source (T-852684; MENTOR GmbH & Co. Präzisions-BauteileKG, Erkrath, Germany) was then adjusted to emit alight intensity less than 30% of the luminescencebrightness but well above noise. This was the case at anLED supply voltage of 1.7 V. This light signal with aspectral maximum at a wavelength of 473 nm was thenused for calibrating the sensitivity of the video camerawhile the LED was in the focal plane.

Finally, the dim red background illumination wasadjusted to a level allowing the recognition of theworm’s body contour but down-regulated to such a lowlevel that background light did not interfere with videoimaging of the luminescent Xashes.

Results

Luminescent sites

Luminescence in E. blomstrandi is conWned to the trunk.In response to extremely strong stimulation, light can beproduced in every trunk segment (Fig. 1). Luminescentlight is emitted from a pair of patches in each of thetrunk segments (Fig. 1). Each of these sources is a roundpatch of luminescent tissue of the dorsal epidermis sur-rounding the base of the dorsal cirrus (Fig. 2). Everysuch patch appears to be composed of a number ofsmaller light sources. Minor accessory light sources arealso found elsewhere in the dorsal epidermis (Fig. 2) andventrally in the parapodial bases. Light emission alwaysremains conWned to the epidermal domains and thusapparently does not originate in secreted material.

Eliciting and observing bioluminescent activity in Eusyllis

In the material included in the present study, lightemission was induced by touching the worms with ametal tool (Fig. 2), by pulling on the wall of the silk

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tubes that the worms construct and inhabit (Figs. 3, 7),or by single or repeated mechanical shocks to the sub-strate (Figs. 4, 5, 6, 10). Electrical stimulation wasabandoned after it was found that considerable voltage(¸5 V) is necessary to overcome the threshold of reac-tion and because many worms under such conditionsautotomized right upon stimulation.

The usual luminescent display of E. blomstrandi isseen by the naked eye as a series of Xashes. Most Xas-hes appear to move along the trunk, but the course and

spatial pattern of the luminescent events in Eusylliswas only revealed through high-speed video records. Inthe present study, we have used video imaging at a fre-quency of up to 100 frames/s to obtain informationabout the sites of luminescence and the kinetics of lightemission: changing brightness, propagation of lumines-cent activity (excitation) by recruitment of additionalsegmental light sources, and fading.

The individual light Xash

We followed the emission of light in whole worms andin posterior trunk fragments. Posterior trunk frag-ments survive for weeks and remain functional withrespect to luminescence. Their patterns of serial Xash-ing appear less modulated than those in whole wormsand therefore play a role in our characterization of thespatio-temporal pattern of light emission. The poster-ior ends that we have used for video imaging had frag-mented the same day that the images were taken.

Upon mechanical stimulation, a Wrst very faint lumi-nescent reaction was observed after a brief delay.Luminescence may start as dim Xashes, for exampleafter a latent period of 165 ms in the case shown inFig. 10. It may, alternatively, start as a Xicker com-posed of asynchronous, spatially incoherent, faintpulses (·10 ms) of luminescent light from single lumi-nous domains, and in one case this Xicker began 116 msafter the worm was touched with a needle. Followingthis brief phase of spatially and temporally rathererratic luminescence, serial Xashing sets in.

Fig. 1 Eusyllis blomstrandi. After extremely strong stimulation, all of the trunk segments may luminesce

Fig. 2 Posterior end of a luminescing, intact Eusyllis blomstran-di. Note the elongate dorsal cirri (dark, Wlamentous contours pro-jecting on either side of the trunk) originating in the middle of thesegmental luminescent domains and the individual segmentalpatterns of small, accessory light sources in the vicinity of themain luminescent domains. In consecutive luminescent Xashes,the same luminescent sites are involved. Stimulation by a touchwith a needle

Fig. 3 Luminescence of Eusyllis blomstrandi elicited by usingforceps (lower left; luminescent light is reXected at its tip) to pullon the silk tube (arrow) which the worm has constructed fordwelling

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Figures 4 and 8 each show a sequence of videoimages taken at 10 ms intervals, illustrating the propa-gation and fading of luminescent excitation during asingle Xash in a posterior fragment and in a whole

worm, respectively. We have processed some of theinformation in such images as shown by the diagramsin Fig. 5 where luminescence of each of the segments isplotted over time. A square unit represents the degreeof luminescent activity of a single segment (verticalaxis) in a 10-ms interval (horizontal axis). Left andright photocyte clusters of a segment are representedby a single square, as both of them with few exceptionsshare the same degree of light-emitting activity. Bright-ness of emitted light is expressed in these diagrams bythree categories: (1) white squares—full light satura-tion in all those pixels that make up the image of oneluminescent site; (2) light grey squares—the majorityof pixels appear saturated; (3) dark grey squares—aminority or none of the pixels saturated. Segments andintervals in which the luminescent system was silent arerepresented by black squares.

A Xash starts with the apparently simultaneous lumi-nescent activity of a group of consecutive trunk seg-ments (Figs. 4, 8). Depending upon the site along thetrunk at which luminescent excitation starts, the lumi-nescent activity expands either posteriorly (Figs. 4, 5, 6,late Xashes in Figs. 7, 9b, c) or anteriorly (Fig. 10, lateXashes) or simultaneously in both a posterior and ananterior direction (amphidromous propagation;Figs. 7, 8, 9a, 10, early Xashes). In posterior fragments,all segments become involved in light emission(Figs. 4, 5, 6). As at the beginning of a Xash, lumines-cence does not simultaneously terminate in all of thesegments: light fading appears as a wave that runs alongthe series of segmental light sources. Such a sequentialcourse of both the start and the fading of light emissionalong the chain of trunk segments is a general patternobserved in the light Xashes produced by Eusyllis.

Fig. 4 Eusyllis blomstrandi. The pattern of luminescence in thecourse of a single Xash [labelled (a) in Figs. 5, 6] in a fragmentcomprising the posterior 29 segments of a 68 segment-long worm.Luminescence starts in the anterior segments (nos. 39–49 on top

in this Wgure) and ends in the most posterior segments of the frag-ment. The video images are taken at 10 ms intervals (seeSect. ”Materials and methods”). Stimulation by mechanicalshocks to the glass support

Fig. 5 Eusyllis blomstrandi. Schematic representation of the seg-mental distribution and intensity of light emission in an early (a),middle (b), and late Xash (c) of a series in a posterior fragmentidentical with that shown in Figs. 4 and 6. A square unit repre-sents one segment (vertical axis) and a time interval of 10 ms (hor-izontal axis). The numbers on the ordinate left and right of thegraphs in this Wgure and in Figs. 6, 7, 9, and 10 symbolize the posi-tions of the individual segments in the trunk in an anterior–pos-terior (left) and in a posterior–anterior (right) direction. Anumbering from the posterior end towards the anterior is includ-ed because the light pulses may start from the posterior-most seg-ment (Fig. 10), and because the total number of luminescingsegments may increase in subsequent Xashes towards the ante-rior, such that the posterior-most segment may become the pointof reference. By estimating the light intensities seen in the origi-nal video images, like those in Figs. 4 and 8, three categories ofemission intensities are symbolized in the graphs by plotting man-ually three grades of shading, from dim light (heavy shade) to fulllight intensity (no shade), as shown in a representing the originalvideo images of Fig. 4

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The following detailed information can be gatheredfrom analysis of the diagrams in Figs. 5, 6, 7, 9, and 10.Excitation, expressed as luminescent activity, quicklypropagates by recruiting segment after segment with adelay of, in some cases, less than ·1 ms/segment. In theexample shown in Figs. 4, 5, and 6, luminescence prop-agates from the anterior towards the posterior endwhere the excitation arrives after 10–20 ms in the earlyXashes of a series and after 40 ms in the late bursts(Fig. 5a, c). Likewise, the Xash fades over a period ofabout 20–40 ms. Faint light emission, or a low level ofexcitation, propagates as faint emission, and strongemission propagates as a strong emission. Thus, coordi-nation of light emission among the segments also per-

tains to quantitative aspects of luminescence. In asingle segment, light intensity increases during an earlyXash and then decreases. In later Xashes, an individualsegment typically reaches full light intensity during theinitial 10 ms whereas fading usually takes 10–40 ms. Asa combined eVect of intersegmental propagation andintrasegmental modulation of light intensity, the lightmaximum may shift during a Xash along the series oftrunk segments (see below).

Flashes in a series

The typical Xash series of E. blomstrandi is a rhythmicdischarge, with a frequency of about 7 Hz and a total

Fig. 6 A Xash series emitted by a posterior fragment of Eusyllisblomstrandi following a series of mechanical shocks to the sup-port (identical material as in Figs. 4, 5). The Xash series was rhyth-mic but asynchronous to the shocks. The Xashes labelled by a, b,

and c are seen as close-ups in Fig. 5. Coordinates and symbols asin Fig. 5. Left margin Drawing of a posterior fragment ofE. blomstrandi

Fig. 7 Diagram showing a Xash series in an intact Eusyllisblomstrandi with 68 trunk segments (see drawing at left margin ofan E. blomstrandi by K. Rehbinder). Stimulation was by pullingon the silk tube (see Fig. 3). Light emission is restricted to the pos-

terior segments, with a gradual recruitment of a number of moreanterior segments. Flash (a) is illustrated by an original video inFig. 8, and Xashes a, b, and c are shown in more detail in Fig. 9.Same animal as in Fig. 3. Coordinates and symbols as in Fig. 5

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duration of about 1.5–2 s (Figs. 6, 7, 10). The rhythmsof Xashing and the quantitative aspects of lightemission of E. blomstrandi are best studied withphotomultiplier recordings and will be treated in alater paper. Here, we concentrate on the contributionof individual segments and on the intersegmental coor-dination of Xashing in both posterior fragments andwhole animals.

A Xash series emitted by a posterior fragment isshown in Fig. 6, and Xash series from intact worms inFigs. 7 and 10. It is evident that (1) Xash frequency Wrstaccelerates and Wnally slows down in the course of aseries, (2) both the light intensity and the duration ofthe Xashes increase and may continue at a high level,(3) a Xash series terminates with a full burst instead ofXash peak heights diminishing continuously towardsthe base line, (4) Xashing in a posterior fragmentinvolves all of the segments, while in whole worms,with exceptions, only a posterior part of the trunk isemitting light, and (5) the site on the trunk where exci-tation starts in a Xash may shift from Xash to Xashtowards a terminal position, e.g. towards the posteriorend, as in the case shown in Fig. 10.

The earliest Xashes in a series may fail to reach fullbrightness in some or in all of the luminescing segments(shown in Fig. 10). Alternatively, Xashes in a series maystart with dim light, and switch to full intensity after a10-ms interval, or in late Xashes the segmental lightsources may commence at full light intensity already inthe Wrst 10 ms interval. Early Xashes last for a shortperiod of time only in an individual segment (·10–60 ms). In this case light production ceases completelybefore the next Xash sets in. In later Xashes, light emis-sion may last longer in the individual segment, e.g.100 ms in total (Figs. 5, 6) or more (Figs. 7, 9).

During a Xash series, luminescent activity may increaseto such a degree that light is produced during the wholeindividual Xashing cycle such that the Xashes tend to over-lap. Flashing at such an elevated level of excitation

appears as a rhythmic increase and decrease of brightnesssuperimposed upon permanent light emission at a lowerlevel (Fig. 7). In extreme cases, e.g. immediately beforeand after fragmentation, all of the light sources luminescecontinuously for seconds at full brightness, in whole ani-mals as well as in posterior fragments (see below). LateXashes in a series typically persist in a state of weak lumi-nescence for an extended period of time (60–80 ms). Thisis best seen in the last Xash of the series in Fig. 7: A termi-nal posterior group of 16–18 segments including the lastsegment remain in a state of dim luminescence beforetheir lights Wnally vanish synchronously.

Luminescent Xashing in whole animals, other than inposterior fragments, is usually restricted to a posteriorpart of the trunk and always includes the terminal seg-ments (Figs. 7, 8, 9, 10). During a Xash series, the group

Fig. 8 Video images of a single Xash out of the series emitted by an intact Eusyllis blomstrandi [Xash (a) in Figs. 7, 9]

Fig. 9 Details of Fig. 7 (Xash series in an intact Eusyllis blomst-randi). Note the increasing duration of the Xashes, the recruit-ment of more anterior segments, the increasing duration of brightlight emission per segment, the delay in the beginning of brightlight emission existing between the more anterior and the poster-ior segments in (b) and (c), and the prolonged emission and dimlight phase typical of the last Xash in a series (c). Same coordinatesand symbols as in Fig. 5. The non-luminescing anterior segments(see Fig. 7) are omitted from this graph

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of segments involved in luminescence expands for-ward, recruiting Xash by Xash new segments, e.g. 18 inthe case of Fig. 7, and 17 in the case of Fig. 10. Recruit-ment towards the anterior terminates somewhere inthe middle or anterior part of the trunk. In late Xashes,some of the anterior segments recruited before maystop luminescing again before the last Xashes of theseries set in. The borderline between the posteriorluminescent and the anterior non-luminescent seg-ments is always distinct, such that a fully luminescentsegment adjoins a completely inactive one, eventhough this borderline during a Xash series repeatedlyjumps from one segment border to another.

Luminescent behaviour following fragmentationof the worm

Eusyllis blomstrandi, like all of the free-moving poly-chaetes, is often subject to loss of trunk fragments in itsnatural habitat. Unlike the posterior fragments, theanterior fragments can fully recover from the loss byregenerating the lost part. E. blomstrandi have beencut into two pieces or, by strong mechanical stimula-tion, were induced to commit autotomy in our experi-ments. Fragmentation is always linked with an outburstof strong luminescence.

Luminescent behaviour displays a striking asymme-try between anterior and posterior fragments(Fig. 11a, b). After autotomy or artiWcial fragmenta-tion, the anterior fragment rapidly departs and aban-dons luminescence after emitting some more lightpulses during a brief period of time (four Xashes in the

case shown in Fig. 11a, b emitted during a period of806 ms). The posterior fragment, on the other hand,Wrst glows for a few seconds extremely brightly andthen continues luminescence in the Xashing mode for aperiod far exceeding the duration of a regular Xashseries, in the case shown in Fig. 11a, b almost 6 s. Theposterior fragments remain responsive to stimulationand capable of luminescent Xashing for weeks despitetheir inevitable starvation.

Discussion

Analysis of the luminescent behaviour of the poly-chaete E. blomstrandi, using high-speed video imaginghas revealed rhythmical Xashing as the basic pattern oflight emission. The kinetics of Xashing is characterizedby co-operative light emission from epidermal lumines-cent domains in a number of segments, by the capacityof the luminescent domains to modulate the intensityof the light emitted, and by coordination of the lumi-nescence patterns among the segments in space andtime. In the typical luminescent response, light emis-sion is restricted to the posterior segments. A divisionof labour and risk is demonstrated by experimentalfragmentation of the trunk when the posterior frag-ment continues to display particularly bright lumines-cence while the anterior fragment with the head andthe feeding apparatus stops emitting light and rapidlymoves away from the site of the damaging encounter.All these phenomena can be viewed in motion pictureon-line in Zörner and Fischer (2006).

Fig. 10 A complete, brief Xashing cycle emitted after a singlemechanical shock (arrow) to the glass support carrying an intactEusyllis blomstrandi of 58 segments trunk length. Note the amp-hidromous propagation of excitation in the early Xashes accom-

panied by a shift of the starting points of the Xashes towards theposterior end. In the later Xashes, recruitment of luminescing seg-ments runs from posterior towards anterior. Same animal as inFig. 3. Coordinates and symbols as in Fig. 5

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Every segment of E. blomstrandi is equipped withpaired clusters of photocytes in the epidermis in thedorsal parapodial base and additional small lumines-cent units. Luminescence is always conWned to theseepithelial sites and thus appears to be strictly intracel-lular. Intermittent light emission might seem to resultfrom repeated cycles of decay and might thus suggestthe requirement for refuelling the light-emitting systemafter every Xash. Such a fuelling problem, however, isobviously not the ultimate reason for the Xashing pat-tern since Eusyllis can produce light continuously forseveral seconds following extreme stimulation. TheXashing mode of light emission, therefore, must haveanother basis and is likely to be functionally relevant.

Light emission in the Xashing mode in contrast witha continuous glow requires rapid intersegmental signal-ling and thus suggests a centralized neural coordinationof luminescent activity. A number of observations sup-port the existence of a central control of luminescencein E. blomstrandi: Both left and right luminescentdomains in a segment start and terminate light emis-sion synchronously. A particularly strong argument isthe high speed at which excitation propagates andfades along the segmented trunk. Light emission maystart in as many as 24 segments “at once,” i.e. duringthe basic unit of time resolution possible with ourequipment (10 ms), and light emission may cease in upto 30 segments during the same time span. From thesedata, a time interval of as little as ·1 ms can be extrap-

olated as a minimum time required for transmitting orsilencing of the excitation from segment to segment.Likewise, the modulation of luminescent brightnesscan propagate among the segments at the same highspeed.

The kinetics of luminescence in Eusyllis also suggestthe existence and activity of a pacemaker controllingthe rhythmic course of Xashing. Rhythmic light dis-charge can be triggered by a unique, arrhythmic stimu-lus, proving the capability of the worm for autonomousrhythmic Xashing. The modulated rhythm of Xashingdoes not appear to be inXuenced by the variable dura-tion of the individual Xashes: Even if the late Xashes lastlonger and tend to overlap, the phase-length allotted tothe single cycle of Xashing does not extend correspond-ingly (Figs. 7, 10). The location of the pacemaker alongthe trunk appears variable: In Fig. 6 and in the late Xas-hes of Fig. 7 the anterior-most luminescent segmentslight up Wrst, suggesting that the pacemaker functionresides in these anterior segments. In Fig. 10, the pace-maker function appears to move during the Wrst WveXashes from segments 37/38 in the Wrst Xash to the last,58th segment in the Wfth Xash and to remain there untilthe end of the Xash series. In the Wrst part of the Xashseries shown in Fig. 7, the pacemaker function consoli-dates at the posterior end before the sense of pacemak-ing reverts in the second part of the series. Thepacemaker function is even divisible: Immediately afterfragmentation, the anterior fragment of Eusyllis still

Fig. 11 a Under the squeezing pressure of a scalpel blade (doublearrow light reXections on the blade) and 525 ms prior to autotomya Eusyllis blomstrandi luminesces simultaneously in all of itstrunk segments. b Luminescent behaviour of the anterior andposterior fragments of E. blomstrandi, same animal as in Fig. 11a,

1,158 ms later and 633 ms after fragmentation. The light emissionhas almost completely vanished in the anterior fragment (topright) while in the posterior fragment luminescence continues for8.37 s after fragmentation, the initial 2.54 s of this period lumi-nescing incessantly at full brightness

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continues rhythmic Xashing for a few bursts (four in thecase shown in Fig. 11a, b) before luminescence becomessilenced, and the posterior fragment, after emittingmaximum light intensity continuously for some seconds,resumes a periodic Xashing activity as well. RhythmicXashing can also be organized in isolated scales andeven in fragments of scales of polynoid polychaetes(“emissions autoentretenues”; Bilbaut and Bassot1977). Thus, the rhythmicity of luminescent discharge inpolynoids depends neither upon an intact segmentalorganization nor an intact central nervous system.

Two sorts of thresholds indicate a central, neuralcontrol of luminescence in Eusyllis. First, a thresholdappears to exist for a Xash event to take place. The lastXash in a series is displayed at full or nearly full bright-ness by a substantial number of the segments beforeluminescence suddenly vanishes. This suggests that anintegral threshold valid for the whole trunk or trunkfragment is no longer being crossed and that it has tobe crossed for a Xash to take place at all. On the con-trary, in the polynoid scale system serial Xashes emit-ted by isolated scales devoid of central nervousconnections decrease continually and exponentially inamplitude with continuing stimulation until completefading of responsiveness (Bilbaut and Bassot 1977).The most anterior of the luminescent segments mayemit light at full brightness while the adjacent segmentfurther to the anterior remains completely dark; never-theless, that adjacent segment may be recruited forbright luminescence in the following Xash. On theother hand, in posterior fragments all of the segmentsparticipate in light emission. This suggests the absenceof a threshold in such fragments while in the intact ani-mal the anterior end may exert a silencing eVect, estab-lishing a threshold in the anterior segments andthereby controlling the expansion of luminescencefrom the posterior to the anterior part of the trunk.The assumed existence of an inhibitory eVect exertedby the anterior end is also supported by the rapid haltto which light emission is brought in the anteriorfragment after the worm has been divided into twofragments.

Facilitation appears to take place in the course of aXash series. This can be concluded from three sorts ofphenomena: Initially, the Xashes appear weak, as mostof the activated segments emit dim light only, whereaslater the activated segments tend to start a Xash emis-sion at full light intensity. Second, the duration of lightemission in a Xash increases during a series. Third, thegroup of luminescent trunk segments initially, Xash byXash, recruits more anterior segments. It is unknownwhether facilitation takes place at the eVector tissue orthe neural level.

In the scale worm system, facilitation is broughtabout by recruitment of previously inactive cells of thescale epidermis as well as by intracellular recruitmentand coupling of the photosomes, the luminescentorganelles (Bassot 1987). Working with free-moving,un-anesthetized worms or worm fragments at low opti-cal magniWcation, we could not yet ascertain a roleplayed by intracellular coupling of intracellular organ-elles in modulating the intensity of segmental lightemission in Eusyllis.

Fatigue, the phenomenon complementary to facili-tation, occurs to a lesser degree than facilitation. It maybecome manifest as a decrease in the number of seg-ments involved in luminescent discharge (Fig. 7) and/or a lowering of light intensity emitted from the indi-vidual segment (Fig. 6). In the case of isolated polynoidscales, fatigue can be attributed to the deWnitiveexhaustion of photogenic material (Bassot 1987). In E.blomstrandi, however, the decrease of intensity thatmay be observed in the later course of a Xash seriescannot be explained by a shortage in the stock of pho-togenic material: Harsh treatment of the worm canwithout delay lead to extended periods of full strengthluminescence and thus reveals the existence of largestocks of photogenic material that would otherwise gounnoticed. Thus, a diVerent explanation must accountfor the fatigue phenomenon observed in Eusyllis lumi-nescence.

We still have no information on the neural organiza-tion of bioluminescence control in syllid polychaetes.The existence of giant Wbres in the ventral nerve cord isa common feature of the polychaetes which allowsrapid communication among the segments and quickreXexes. SpeciWc information on the structure andfunction of this system in relation to syllid biolumines-cence, however, is lacking.

Our present paper on the kinetics of Xashing lumi-nescence in E. blomstrandi is the Wrst on this subject,with the exception of a brief description of Xashing bio-luminescence in the closely related syllid P. pulligera(Bassot 1979). Polychaete bioluminescence has, how-ever, been extensively studied in the very special case ofpolynoid scales that are easily shed upon irritation ofthe worm and can emit bright luminescence. The lim-ited level of emitted luminescent light and the resultingtechnical problems in video imaging were overcome inthis system by using artiWcially induced epiXuorescentlight, as the light-producing organelles in polynoidsbecome Xuorescent while luminescing. Detailed knowl-edge has been gathered in this system on the histologi-cal and organelle structure of the luminescent tissueand its nerve equipment as well as on many kineticparameters of luminescent activity (Bassot 1966, 1979,

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1987; Bassot and Bilbaut 1977a, b; Bilbaut and Bassot1977; Pavans De Ceccatty et al. 1977; Herrera 1979;Hastings et al. 1987). After this impressive start ofresearch on polychaete luminescence in the polynoidscale system, we now expect that working with theintact Eusyllis, possessing its complete neuronal cir-cuitry and behavioural integrity, will expand our under-standing of how luminescence in polychaetes isoperated and controlled and will justify the establish-ment of a diVerent experimental animal. Self-mutilationafter electrical stimulation in Eusyllis indicates thatmechanical instead of electrical release of luminescenceis preferable for the study of typical luminescent pat-terns and raises the question whether the occurrence of“atypical” patterns of luminescent Xashing in isolatedscales (Bilbaut and Bassot 1977) might be artefacts dueto electrical shocking. Moreover, the use of modernequipment gives us the possibility of using natural lightfor imaging of the luminescent discharges in E. blomst-randi, whereas equipment-induced Xuorescent light hadto be used for technical reasons in the imaging of poly-noid scale luminescence (Bassot and Bilbaut 1977b).

The functional signiWcance of luminescent behav-iour in marine animals has for a long time intrigued sci-entists, and the numerous explanations (Morin 1983;Hastings and Morin 1991) still rest on little experimen-tal evidence. In some cases, the plain observation isvery suggestive of certain functions, as in the “Bermu-dian Wreworm,” O. enopla. The ripe females in this spe-cies give oV a luminescent secretion which, accordingto Weld observations (Fischer and Fischer 1996),attracts ripe males. Morin’s classiWcation ascribes adeterrent function to bioluminescent Xash signals andan attracting function to luminescent “glow” (continu-ous emission of ¸2 s).

In many trials, we have made the consistent observa-tion that fragmentation of a Eusyllis is immediately fol-lowed by an excessive, long-lasting luminescent displayof the posterior fragment and a rapid evanescence oflight emission in the anterior fragment. Immediatelyfollowing fragmentation, luminescence of the posteriorfragment is a bright glow slowly changing into a Xashseries of unusually long duration, and light emissioncontinues to be easily elicited by mechanical stimulationin posterior fragments as long as they survive. Thisobservation suggests that the behaviour of the posteriorfragment is designed to attract the attention of potentialpredators while the non-luminescent anterior fragmentcan meanwhile escape in the dark. This interpretationcan be extended to the luminescence pattern in intactworms: Light emission is usually conWned to the poster-ior part of the trunk and might distract the attention ofa predator from the anterior end of the prey.

The luminescent behaviour of E. blomstrandistrongly suggests a beneWcial eVect: Only the anteriorfragment is capable of regenerating the lost part andcan survive as a potentially reproductive member ofthe population while posterior trunk fragments maysurvive for a while but are incapable of regeneratingthe lost head and feeding apparatus (personal observa-tion; Boilly 1961). Bioluminescence of the posteriortrunk or trunk fragment in an antagonistic encounterwould serve the purpose of a decoy or an alerting sig-nal. The discharge of luminescent light by autotomizedscales of polynoids has been interpreted to have thesame function (Herrera 1979). Since E. blomstrandi isreadily available and we know its luminescent behav-iour and how to handle the worms, we can now think ofobservations strictly demonstrating the validity of thesuggested beneWcial eVect. Testing the validity of ourhypothesis in an experimental predator–prey encoun-ter would, however, require the knowledge of thepotential predators in the littoral habitat of Helgoland.

Acknowledgments This research work has been supported by agrant from Deutsche Forschungsgemeinschaft/Bonn (Fi 182/21-1). The authors are very grateful to Dr H.-D. Franke/Helgolandand the divers of the Biologische Anstalt Helgoland/Alfred We-gener Institute for Polar and Marine Research Bremerhaven(AWI) for providing E. blomstrandi. The same institution hasalso for years generously provided the basic element of ourpolychaete research, natural seawater from the open North Sea.Dr K. Wiese/Hamburg, Germany, with his scientiWc and technicaladvice, helped to get the present work started, and Drs M.Dambach/Köln, Germany and P.C. Schroeder/Pullman, USAcritically read and improved the manuscript. HAMAMATSUInc. (Dr W. Gräwe, Geldern, Germany) repeatedly suppliedsophisticated instruments for video imaging of the biolumines-cence. Dr W. Stickan, Dipl. Ing. K. Seack and Dipl. Ing. J.Kaeding at the IWF Wissen und Medien at Göttingen/Germanymade their competence and skills available to our video imagingproject.

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