Evidence of Luminous Bacterial Symbionts in the Light Organs Of
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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 2591-8 (1991) Evidence of Luminous Bacterial Symbionts in the Light Organs of Myctophid and Stomiiform Fishes DAVID FORAN Department of Biology, The University of Michigan, Ann Arbor, Michigan 481 09 ABSTRACT The myctophids and stomiiforms represent two common groups of luminous fishes, but the source of luminescence in these animals has remained undetermined. In this study, labeled luciferase gene fragments from luminous marine bacteria were used to probe DNA isolated from specific fish tissues. A positive signal was obtained from skin DNA in all luminous fishes examined, whereas muscle DNA gave a weaker signal and brain DNA was negative. This observa- tion is consistent with luminous bacteria acting as the light source in myctophids and stomiiforms and argues against the genes necessary for luminescence residing on the fish chromosomes. To confirm the location of this signal, a bacterial probe was hybridized in situ to sections of a stomii- form. A strong signal was generated directly over specific regions of the fish light organs, whereas no signal was found over other internal or epidermal tissues of the fish. Taken together, these data provide the first indication that luminous bacterial symbionts exist in myctophids and stomii- forms and that these symbionts account for luminescence in these fishes. Luminous fishes make up a major portion of the photophores, small, often innervated light organs, oceans' mid- and deep-water fauna. However, in which are generally found in one or two ventral only a fraction of these is the mechanism of lumi- rows on the skin and may occur elsewhere on the nescence well understood (reviewed by Harvey, body. Further, some stomiiforms have a luminous '52; Herring and Morin, '78; Hastings and Neal- barbel, and both groups may contain luminous son, '81; Hastings, '83). Where it is well character- patches or scales (e.g., Harvey, '52; Jorgensen and ized, as in species of Physiculus, the Ceratioidae, Munk, '79). This array of light organs can be used and the Anomalopidae, among others, fish lumi- as lures, for schooling purposes, or as defense nescence depends on a symbiotic relationship be- mechanisms; light organs may also be secondary tween the animal and one of a number of lumi- sexual characters. nous bacterial species that not only exist in the In spite of the vast quantity of research directed light organs of the fish but are also common in towards myctophids and stomiiforms, there are the oceans' waters (Harvey, '52; Hastings and still few or no firm results regarding their method Nealson, '81). Such bacterial symbionts have been or methods of luminescence. Extensive micro- confirmed in cultures generated from light or- scopic studies of the light organs of these fishes gans, by microscopy (e.g., Bassot, '66); through give no indication of the presence of symbiotic assays for the enzyme responsible for lumines- bacteria (e.g., Bassot, '66; Herring and Morin, '78; cence, bacterial luciferase (Leisman et al., '80); Jorgensen and Munk, '79). Further, a luciferase and in one case by molecular techniques (Hay- has not been isolated from these fishes, nor from good and Cohn, '86). In these fishes, light emis- any fish that does not harbor luminous bacteria. sion from the constant bacterial glow is controlled Some positive results have been obtained by by mechanical means, such as a lid pulled over assaying preparations of luminous fishes for the the light organ (Photoblepharon). luciferase of coelenterates (reviewed by Hastings, This is not the case, however, in two common '831,but this assay is positive in a broad range of groups of luminous fishes, the myctophids and organisms (Hastings, '83) and no successful at- stomiiforms, where luminescence appears to be tempts have been made to isolate the enzyme. under physiological control and ranges from a slowly propagated glow to flashes occurring sev- Received March 2, 1990; revision accepted September 12, 1990. David Foran's present address is Ludwig Institute for Cancer Re- eral times per second (Harvey, '52). Luminescence search, 687 avenue des Pins ouest, Montreal, Quebec, Canada H3A in the myctophids and stomiiforms arises from 1Al. 0 1991 WILEY-LISS, INC. 2 D. FORAN The general inability to identify a mode of lu- 8, 100 mM EDTA; and submerged in 1-2 ml of minescence in myctophids and stomiiforms (as the same buffer. For muscle, remaining skin was well as some other luminous fishes) has led to a scraped away and internal organs and head were default assumption that the fishes are themselves removed, and the tissue was rinsed and sub- responsible for their luminescence, with the genes merged in 2-4 ml of buffer. When possible, the encoding luciferase residing on the fish chromo- skull was cleaned of all surrounding tissues and some (e.g., Hastings and Nealson, '80; Smith, '89). cut open from the spine, exposing brain tissue. From this, two hypotheses can be proposed: 1) This was removed and placed in buffer. that these fishes have evolved their own method Tissues were ground for 5-20 sec at full speed of luminescence (presumably independently as with a Tek-Mar Tissumizer. Proteinase K and the myctophids and stomiiforms are not closely Sarcosyl were added to 100 pg/ml and 0.5%,re- related) or 2) that these fishes have incorporated spectively, and samples were incubated at 55°C the genes necessary for luminescence via lateral for 3 hr; 500 pg/ml of ethidium bromide was gene transfer from luminous bacteria, circum- added to each sample, followed by 1 g/ml of venting the need for a symbiont. However, nei- cesium chloride. Solution densities were deter- ther of these possibilities can be favored, in that mined and adjusted to 1.54-1.56 g/ml if needed. the genes encoding luciferase have not been iso- Samples were centrifuged in a Beckman VTi 65 lated. rotor at 55,000 rpm, 21"C, overnight. DNA was Recently, a number of groups have succeeded in isolated, and a second centrifugation step was cloning into Escherichia coli the bacterial genes performed using a Beckman SW 60 rotor at necessary for luminescence (Belas et al., '82; Cohn 36,000 rpm, 21"C, overnight. The DNA band was et al., '83; Engebrecht et al., '83; Delong et al., again isolated, extracted repeatedly with water- '87). In luminous bacteria, luciferase is a dimeric saturated butanol to remove the ethidium bro- protein encoded by two linked genes termed Lux mide, and dialyzed extensively against 10 mM A and Lux B (reviewed by Hastings et al., '85). Tris, pH 8, 1 mM EDTA. The availability of these DNAs allows their use as probes to determine if homologous genes might Construction of bacterial and fish clones to be be found in the tissues of myctophids or stomii- used asprobes forms. If these fishes have incorporated the bac- The Vibrio fischeri Lux clone pJE205 (Enge- terial Lux genes into their genomes, one would brecht et al., '83) was obtained from K. Nealson. expect to obtain a positive signal from all fish It was digested at the sites indicated in Figure 1 tissues examined. If previously undiscovered lu- and ligated into Bluescript vector DNA (Strata- minous bacteria are present in the light organs of gene Cloning Systems) using published protocols these fishes, one would expect a positive signal (Maniatis et al., '82). The ligation mixture was only from those tissues containing light organs used to transform competent E. coli cells (XL1 (the skin, and muscle to the extent the light or- blue, Stratagene). Plasmid DNAs from trans- gans are embedded in it) and a negative result formed bacteria were isolated by the alkaline ly- from more internal tissues. Finally, if the fishes sis method (Maniatis et al., '82). In a similar man- have evolved their own method of luminescence, ner, fragments of fish DNA were cloned for use as one would expect a negative result from all fish positive controls and to help quantitate the rela- tissues. tive amount of DNA in a given lane on fish blots. For use as probes, clones were digested with MATERIALS AND METHODS restriction enzymes that left only 20-50 bp of vec- tor DNA attached to the inserts, just enough so Isolation of fish genomic DNAs that vector sequences complementary to sequenc- Fish samples were obtained by Karsten Hartel ing primers remained. Inserts were then electro- from the western North Atlantic. Animals were phoresed through low-melting-point agarose frozen on board ship and sent to Michigan, where (BRL) and isolated according to manufacturer's they were stored at -60°C until use. Once each instructions. Approximately 100 ng of the iso- fish was identified for family, genus, and/or spe- lated fragment was heated to 100°C for 5 min. An cies, DNA was isolated from skin, muscle, and excess of primers complementary to both strands brain. Over wet ice, skin from each animal was was added and the DNAs were radiolabeled by teased away from underlying muscle; immedi- primer extension (Maniatis et al., '82). In the case ately rinsed with cold, sterile, 100 mM Tris, pH of most probes, the primers utilized were the "SK" LUMINOUS BACTERIA IN MYCTOPHIDS AND STOMIIFORMS 3 and “KS” primers of Stratagene. For the pSH700 was completely free of contamination by other tis- probe, primers were synthesized that were com- sues. In this way, total DNA was isolated from a plementary to sequences internal to the restric- number of myctophids and stomiiforms represent- tion sites. ing the genera Photostomias, Chauliodus, Sto- mias, Gonostoma, and Argyropelecus. Blotting and probing of fish DNAs DNA samples were digested with a variety of Survey of fish tissues restriction enzymes and electrophoresed through The luciferase clone pJE205, isolated from Vib- 0.8% (for enzymes recognizing six base sites) or rio fischeri, has been characterized (Engebrecht et 1.2% (four base sites) agarose gels.