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Notice: ©1992 Springer. This manuscript is an author version with the final publication available at http://www.springerlink.com and may be cited as: Herring, P. J., Widder, E. A., & Haddock, S. H. D. (1992). Correlation of bioluminescence emissions with ventral in the mesopelagic veranyi (Cephalopoda: ). Marine Biology, 112(2), 293‐298. doi:10.1007/BF00702474

I I Marine Biology 112, 293- 298 (1992) Marine

lntemattonat dournal llfSB~~7/ In on life Oceans B-Iology J--_--- () and Coastal Waters JJ-)P\J~ - © Springer-Verlag 1992 J Correlation of bioluminescence emissions with ventral photophores in the mesopelagic squid Ahralia veranyi (Cephalopoda: Enoploteuthidae)

P. J. Herring I, E. A. Widder 2 and S. H. D. Haddock 3

1 Institute of Oceanographic Sciences, Deacon Laboratory, Brook Road, Wormley, Godalming GU8 5UB, Surrey, England 2 Harbor Branch Oceanographic Institution, 5600 Old Dixie Highway, Fort Pierce, Florida 34946, USA 3 Marine Science Institute, University of California, Santa Barbara, California 93106, USA

Date of final manuscript acceptance: September 26, 1991. Communicated by 1. Mauchline, Oban

Abstract. Specimens of the squid Abralia veranyi were spinosus and Systellaspis debilis is spectrally different collected off the Bahamas in 1989. The bioluminescence from that emitted by the photophores ofthe same species of the ventral photophores was recorded on videotape at (Herring 1983, Latz et al. 1988), and in many the 11 to 12°C and 24 °C, in conjunction with measurements emission spectra are determined, in part, by the effects of of the spectral emission at the same temperatures. At accessory optical structures such as filters (Denton et al. 11°C, the spectrum was unimodal, peaking at 490 nm 1985). with a narrow bandwidth. At the higher temperature a Reversible spectral changes in the bioluminescence shoulder at about 440 nm appeared in the emission spec­ emission ofa single individual have rarely been reported; trum. The short, bright flashes from the subocular pho­ the most detailed examples are those of the tophores had a broader bandwidth and a shorter wave­ sp. and (Young and length emission maximum (475 nm) than that of the Mencher 1980, Young and Arnold 1982). These authors ventral photophores. Video recordings at the two tem­ suggested that the spectral changes observed (in response peratures showed no changes in either the identities ofthe to changes in ambient temperature or light intensity) re­ luminescing photophores or their relative intensities. One sulted both from recruitment of different photophores structurally distinct type of was not illumi­ and from changes in the spectral emission of at least one nated. We conclude that the observed spectral changes class of photophore. were produced within one group of photophores, and We have recorded the ventral bioluminescence of the that recruitment ofthe unilluminated photophores would related squid Abralia veranyi on videotape and analysed produce an additional spectral component, previously the contributions of different photophores at different reported from another species of Abralia (A. trigonura). temperatures, while monitoring the associated spectral changes. The spectral changes we observed were not asso­ ciated with the recruitment of different classes of pho­ tophore. Introduction Certain species of bioluminescent animals are known to be able to produce different colours of light emission. In Materials and methods most of the cases so far investigated, this is achieved by the employment of different photophores, each of which Specimens of Abralia veranyi were obtained on a cruise of R.V. has a characteristic spectral emission. In some deep-sea "Edwin Link" in the Bahamas in November 1989. Squids were fishes the suborbital photophore produces red light, captured either by dipnetting at the surface or by capture at depth whereas the postorbital photophore produces blue light with the Johnson-Sea-Link submersible. They were brought into the (Widder et al. 1984). Among terrestrial arthropods the shipboard laboratory, where they usually emitted a spontaneous ventral glow. The most detailed observations were made on a spec­ red and green luminescence of the "railroad worm" imen (37.5 mm dorsal mantle length) captured at a depth of 602 m Phrixothrix sp. and the elaterid beetle Pyrophorus plagio- on 16 November at 26° 4.3'N, 77° 34.3'W The ambient temperature phthalmus similarly derive from different photophores at this depth was 12.5 °C. The morphology ofdifferent photophores (McElroy et aI., 1974). Even in cases where specific pho­ from this specimen was examined by embedding a small area of the tophores are not identifiable, different areas of the body ventral mantle in Taab (Reading, UK) premix medium resin. Thick may produce different colours of bioluminescence, as in ("" 2 urn) serial sections were cut on a Huxley Ultratome with glass knives and stained with 1% toluidine blue in 1% borax. the green and blue luminescence of the cnidarian Umbel­ An intensified video camera (ISIT Dage 66) was used to record lula huxleyi (Herring 1983, Widder et al. 1983). The secre­ the bioluminescence on !" (12.7 mm) video tape. Computer image­ tory bioluminescence of the decapod shrimps Oplophorus analysis ofphotophore patterns was carried out using a Megavision 294 P.J. Herring et al.: Bioluminescence of Abralia

1024XM system . A photograph of the formalin-preserved specimen that were not brightly luminescent could often be stimu­ was scanned into the computer system using a Hewlett Packard lated by briefexposure to the illumination ofa hand-held ScanJet Plus scanner for comparison with digitized video frames of flashlight. Luminescence was mostly of relatively con­ bioluminescence. Images of Abralia veranyi at 12 and 24 °C were compared with each other and with the digitized photograph of the stant intensity and any changes that did take place were preserved specimen using geometric correction algorithms to cor­ gradual. Any manipulation of the squid was liable to rect for scale differences and geometric distortions between images . induce a very rapid and bright flash from one or more of Comparisons ofimages were made by subtraction. Possible intensi­ the subocular photophores (usually the large posterior ty or size differences between emitting photophores at the two organ). The pattern of visible mantle photophores temperatures were examined using object analysis algorithms as changed regularly with the respiratory pulsations of the well as by real-time pseudocolour analysis ofvideo playback. Biolu­ minescence images were enhanced and sharpened prior to pho­ mantle musculature (Fig. 1). This pattern change is prob­ tographing digitized images from a high-resolution video monitor. ably exaggerated in the experimental situation by virtue Bioluminescence spectra were measured with an EG & G Prince­ of the fact that the squid is unnaturally flattened against ton Applied Research Model 1460 optical multichannel analyser the bottom of the dish during the inhalant pulse. The (OMA) using a linear array detector consisting of 700 intensified respiratory pulse rate at 24°C was substantially higher photodiodes (Model 1420 detector). Details of the OMA operation than that at 12"C. and calibration have been described by Widder et al. (1983). Specimens were maintained in a glass crystallizing dish in sea water at the experimental temperature and mounted above the vid­ Spectral measurements eo camera, which therefore viewed the squids from below. The bioluminescence of the ventral mantle was passed through a quartz Flashes from the subocular photophore (only measured glass-fibre optic of 5 mm diameter to the collection optics of the at 24 °C) had a unimodal emission maximum at OMA. Water-temperature changes were achieved by changing the ~475 nm, with a full width at half maximum (FWHM) sea water in the dish rather than by transferring the specimen . A short ( - 5 min) period of acclimatization preceded each set of ob­ of 82 to 83 nm (Fig. 2A). The array of ventral mantle servations. photophores at 24°C had a Am • x at 489 to 492 nm and an FWHM of47 to 66 nm, with a distinct shoulder at about 440 nm. At 11°C there was no short-wavelength shoul­ Results der, }'m.x was between 487 and 494 nm and FWHM was 54 to 75 nm (Fig. 2 B). The main distinction between the Visual observations two temperatures was the appearance of the short-wave­ In most cases, Abralia veranyi was spontaneously lu­ length shoulder at the higher temperature. This spectral minescent when brought into the dark room. Individuals change was easily and repeatedly reversible.

Fig. 1. Abralia veranyi (life size) at 12°C. (A) Photophore pat­ tern with mantle contracted; (B) 10 video frames later, mantle ex­ panded; (C) 10 frames later, mantic contracted; (D) 10 frames later, mantle expanded P.1. Herring et al.: Bioluminescence of Abralia 295

A

350 400 450 550 600 650 700 Wavelength (nm)

B

Fig. 3. Abralia veranyi ( x 1.3) at 12°e (lower) and 24°e (upper). Note extr a rows of later al tentacular photophores in lower video­ 350 400 450 500 550 600 650 700 frame. This was also seen in some frames at 24"C, and was probably Wavelength (nm) a function of tent acle orientation Fig. 2. Abralia veranyi. Spectral distribution of biolum inescence emission of (A) posterior ocular photophore at 24°e and (B) ven­ tral mantle at 11 and 24°e to 6, and smaller, much more numerous ones. The larger photophores are further separable into refractive and non-refractive types, each horizontal band being com­ Video recordings of photophore illumination posed of one type only. The smaller photophores are less regularly arranged and also comprise two visibly differ­ Images of the pattern of photophores illuminated at 12 ent types, one of simple appearance and with an opaque and 24 °C are shown in Figs. 3 and 4. The pattern is centre, the other of more complex appearance and with a fundamentally similar, with the minor visible differences clear core. Apart from their size, there is nothing to dis­ ascribable to differences in orientation and intensity of tinguish the simple small photophores from the non-re­ emission. The image analysis of the patterns at the two fractive large ones. These two types were never visibly temperatures confirmed that precisely the same set of illuminated. photophores were illuminated. Both large and small pho­ A representative portion of the anterior mantle bear­ tophores were involved, the large ones giving an impres­ ing some of each type of photophore was removed, the sion of transverse brighter bands. The equivalent pho­ photophores sectioned, and their structures compared tophores can be identified on the preserved specimen with the three types of photophore ("simple", "lensed", (Fig. 5). There was no quantifiable difference in the rela­ and "filtered") described in the Abralia by Young tive intensities of the different sizes of photophores at the and Bennett (1988). The illuminated large photophores two temperatures. matched the "len sed" photophores, as their refractile na­ One clear feature of the comparison between the video ture suggested. The illuminated small photophores were images and the anatomical distribution of the pho­ the "filtered" type, with a complex set of internal reflec­ tophores was that a substantial number ofboth large and tive elements. The large and small unilluminated pho­ small photophores were never illuminated (Fig. 5). tophores were not separable except on the basis of size, and matched the description of the "simple" pho­ tophores. Photophore morphology and pattern The distribution ofthe different types in an area ofthe ventral mantle, measuring 8 x 13 mm in the preserved The ventral mantle alone bore> 550 photophores in the specimen, is shown in Fig. 6. This area contained a total specimen examined in detail (Fig. 6). These are divisible of251 photophores; 162 were illuminated (150 "filtered" into large photophores, arranged in transverse rows of 4 and 12 large "lensed") and 89 were not, when counted 296 P.1. Herring et al.: Biolum inescence of Abralia

Fig. 4. Abralia veranyi ( x 2.7). Mantle at t 2°C (A) and 24 °C (B). The comma shape of the photophores in (B) is due to fact that mantle was moving so fast at the higher temperature that no frame was completely unblurred at high magnification

Fig. S. Abralia veranyi ( x 2). Comparison ofbioluminescence emission with photophore pattern on mantle at t 2"C. (A) Mantle ofpreserved specimen; (8) with superimpo sed photophore bioluminescence; (C) bioluminescence pattern alone (same pattern as Fig. 4A)

from a representative video frame at 12°C. The unillumi­ lensed and filtered. There was no difference in the relative nated photophores consisted of 14 large "simple" pho­ intensities of the two types at the two temperatures. Nev­ tophores, 65 small "simple" and 10 "filtered". ertheless, at the higher temperature an additional spectral shoulder was invariably present. The conclusion to be drawn from these data is, therefore, that the spectral Discussion change occurs within one or both of the photophore types. The video data show clearly that in Abralia veranyi the The question then arises as to which photophore is same set of photophores was functional at both 12 and responsible for the spectral change, and how it is 24°C. This set was made up of two morphological types, achieved. Ifthe change is produced at the molecular level, P.I Herring et al.: Biolumine scence of Abralia 297

spectral change, namely that spectrally selective elements B in the accessory optical structures change in response to temperature change. Young and Arnold (1982) identified .* .': -,*.: .. ...* the proximal reflector and the axial stack interference-fil­ '* •• •• * ( ·0 o. o· ....o· ter as possible sites of such change. The proximal reflec­ • ..,. .. ..• * •• tor is composed of concentric layers of collagen rods •• * *.*. • *.* ...... * • arranged in different orientations. The axial stack is *. 0.*. -.*. 0 made up of multiple stacks of reflective platelets, and .. * * • *- * * · .. minor changes in their separation could change the opti­ r. •·..,. _. •• •• ..,. • 0 *. * mally transmitted wavelengths. 0.·0.:0·0 Recent studies on the mechanisms of physiological .. -*. ... '* • '* '* • * ••• colour change in squid iridophores have shown that the o . ••• *-...... reflecting platelets them selves can rapidly change both .. ** _**0.* *. * •• -* • • • '* •• their composition and thickness (Cooper et al. 1990). • ... ** _* 0 These changes affect the optical properties of the platelet ··*0'*.. :0 ....0·- stacks and , hence, their reflective colours. Iridescent fish­ e ••••• * ••• * •• '* 0 ,*... .. es can achieve the same effect by altering the thickness of · :.-"e*-*-"- the cytoplasmic layers between the reflecting crystals of • .><.. * guanine (Lythgoe and Shand 1982). If this is indeed the mechanism ofspectral change in Abralia veranyi, then the o Lensed ON * large simple OFF change can be assumed to occur in the "filtered" pho­ • FiIt.red ON"'" Small simple OFF tophores alone. If, on the other hand, the collagen reflec­ o Filtered OFF tor can also change its internal organization [as Young and Arnold (1982) speculated], this could contribute to spectral changes within all the photophore types, since all of them have similar proximal reflectors. We believe that Fig. 6. Abralia veranyi . (A) Ventral view of preserved individual the spectral changes are explicable without such a contri­ (dor sal mantl e length 37.5 mm) showing 8 x 13 mm oblong area in bution. which the distribution of different photophore types was mapped; The emission spectra recorded by us displayed rela­ arrow indicates large posterior subocular photophore beneath left tively small changes when compared to those reported by eye. (B) Distribution ofdifferent photophore types in oblong in (A); key indicates position of each type and whether or not illuminated Young and Mencher (1980) and Young and Arnold (1982) for Abralia trigonura. The first account described three different spectral components, one at 440 nm, one no inferences can be drawn from the photophore mor­ at 470 to 480 nm, and one at 536 nm. The 470 to 480 phology. There is a precedent for reversible spectral component was dominant at 6°C, while the other two changes induced by temperature in one strain (Y-1) ofthe components appeared at 25°C. Three similar compo­ bacterium Vibrio fischeri (Ruby and Nealson 1977, Leis­ nents were noted in the second account, but the change man and Nealson 1982, Eckstein et al. 1990). In this from 472 nm to 536 nm was continuously variable (and strain, the in vivo spectral emission has both a blue and interpreted as a spectral shift within one class of pho­ a yellow component. At ~ 25 DC the shorter-wavelength tophores), while the 440 nm peak showed a gradual in­ (blue) component predominates, wherea s at <20°C the crease , and was interpreted as a second class of pho­ longer wavelength (yellow) component of the emission is tophores coming into action. The spectral changes were the greater. This is interpreted largely as emission from considered to be a means whereby the squids matched an associated yellow fluorescent protein, through an en­ their counterillumination to both moonlight in the warm ergy transfer process whose efficiency declines with in­ surface waters at night and to downwelling sunlight at creasing temperature, although other processes are also depth by day. involved (Eckstein et al. 1990). The same process could In Abralia veranyi, we observed the 440 nm emission apply in Abralia veranyi, if the assumption is made that without any additional photophores being illuminated. the low-temperature spectrum is wholly produced by effi­ We therefore ascribe this change to internal events within cient energy transfer to a fluor with the same spectral the photophores. We did not observe any 536 nm contri­ .. properties as the observed bioluminescence. The appear­ bution and conclude that this component derives from ance of the shoulder at higher temperature would then the " simple" photophores, which were never illuminated indicate a reduction in energy-transfer efficiency, and the in our specimens . We therefore agree with Young and appearance of a fundamental 440 nm emission. In this Mencher (1980) and Young and Arnold (1982) that spec­ case, the photogenic crystalloids at the base of the pho­ tral changes in Abralia spp. are the consequence both of tophore (Young and Bennett 1988) should contain a fluo­ additional photophore class recruitment and intra-pho­ rophore with the same emission spectrum as the low-tem­ tophore changes, but differ in the interpretation of the perature bioluminescence. There is no information on roles of different classes of photophore. Our data are this point. limited in that they deal only with the effects of tempera­ The complex reflective elements in the filtered type of ture. Young and his colleagues based their conclusions on photophore provide an alternative hypothesis for the more extensive data, including the effects of high light 298 P.1. Herring et aI.: Bioluminescence of Abralia intensity, but did not have the video comparisons that are Literature cited available to us. Our suggested correlation between a 536 nm component and the unilluminated "simple" pho­ Cooper, K. M., Hanlon, R. T., Budelmann, B. U. (1990). Physiolog­ tophores remains experimentally untested. However, pre­ ical color change in squid iridophores. II. Ultrastructural mech­ anisms in Lolliguncula brevis. Cell Tissue Res. 259:15-24 liminary data from the related squid Watasenia scintillans Denton, E.1., Herring, P.1., Widder, E. A., Latz, M.1., Case, 1.F. do indicate a correlation between temperature-deter­ (1985). The roles of filters in the photophores of oceanic animals mined green bioluminescence and the green fluorescence and their relation to vision in the oceanic environment. Proc. R. of particular photophores (Professor Y. Kito personal Soc. (Ser. B) 225: 63-97 communication). Eckstein, 1.W., Cho, K. W, Colepicolo, P., Ghisla, S., Hastings, The ocular photophore flashes had a broader band­ 1.W, Wilson, T. (1990). A time-dependent bacterial biolumines­ cence emission spectrum in an in vitro single turnover system: width and a shorter wavelength emission maximum than energy transfer alone cannot account for the yellow emission of that of the ventral mantle at 11 to 12 "C. The ocular VibriofischeriY-1. Proc. natn. Acad. Sci. U.S.A. 87: 1466-1470 photophores lack the interference reflector (axial stack) Herring, P.1. (1983). The spectral characteristics of luminous ma­ in the aperture of the "filtered" photophores, and we rine animals. Proc. R. Soc. (Ser. B) 220: 183-217 believe that their spectral emission probably corresponds Herring, P.1. (1988). Luminescent organs. In: Trueman, E. R., to that of the "filtered" photophores before the lumines­ Clarke, M. R. (eds.) The . 11. Academic Press, San Diego, p. 449-489 cence has passed through the axial stack. Their broad Latz, M. I., Frank, T. M., Case, 1.F. (1988). Spectral composition of bandwidth emission is not a good match of downwelling bioluminescence ofepipelagic organisms from the Sargasso Sea. daylight and, as we observed, is probably used more often Mar. BioI. 98: 441-446 in a defensive or "startle" mode. Similar responses have Leisman, G., Nealson, K. H. (1982). Yellow light emission in lumi­ been reported from certain ocular photophores of the nous bacteria: purification and properties of a yellow fluores­ related squids Pterygioteuthis microlampas and P. giardi cent protein. In: Massey, Y., Williams, C. H. (eds.) Flavins and flavoproteins. Elsevier, New York, p. 383-386 (Young et al. 1982, Herring 1988). Lythgoe, 1.N., Shand, 1. (1982). Changes in spectral reflexions from These data do not resolve the question of the function the iridophores of the neon tetra. 1. PhysioI. 325: 23- 34 of the two sizes of"simple" photophore. There is nothing McElroy, W D., Seliger, H. H., DeLuca, M. (1974). Insect biolu­ to suggest that their spectral contributions are different. minescence. In: Rockstein, M. (ed.) The physiology of Insecta. As the large ones occur in horizontal rows which alter­ II. Academic Press, New York, p. 411-460 nate with similar rows ofthe large "lensed" photophores, Ruby, E. G., Nealson, K. H. (1977). A luminous bacterium that it is possible that they may be employed together to emits yellow light. Science, N.Y 196: 432-434 Widder, E. A., Latz, M. I., Case, 1.F. (1983). Marine biolumines­ provide a banding pattern of luminescence with a func­ cence spectra measured with an optical multichannel detection tion quite separate from counterillumination. Further system. BioI. Bull. mar. bioI. Lab., Woods Hole 165: 791-810 observations will be necessary on these fascinating ani­ Widder, E. A., Latz, M.1., Herring, P.1., Case, 1.F. (1984). Far-red mals, both to establish the complete functional ranges of bioluminescence from two deep-sea fishes. Science, N.Y 225: their different photophores and to unravel the full com­ 512-514 plexity of their bioluminescent behaviours. Young, R. E., Arnold, 1.M. (1982). The functional morphology of a ventral photophore from the meso pelagic squid, Abralia trigonura. Malacologia 23: 135-163 Young, R. E., Bennett, T.M. (1988). Photophore structure and evo­ lution within the Enoploteuthinae (Cephalopoda). In: Clarke, Acknowledgements. We are most grateful to Dr R. E. Young for his M. R., Trueman, E. R. (eds.) The Mollusca, 12. Academic Press, constructive comments on the text, to Dr M. Vecchione for identi­ San Diego, p. 241-251 fying our specimens, and to S. Groves for help with the spectral Young, R. E., Mencher, F. M. (1980). Bioluminescence in measurements. This work was supported by grants from the At­ mesopelagic squid: diel color change during counterillumina­ lantic Foundation and from the Office of Naval Research to E. A. tion. Science, N.Y. 208: 1286-1288 Widder (N00014-90-J1819) and to 1. F. Case (N00014-89-J1736 and Young, R. E., Seapy, R. R., Mangold, K. M., Hochberg, F.G., Jr. N00014-84-0314). Harbor Branch Oceanographic Institution Con­ (1982). Luminescent flashing in the midwater squids Pterygio­ tribution No. 871 teuthis microlampas and P. giardi. Mar. BioI. 69: 299- 308