Smaller Research Contributions 145

Bacterial symbionts in the luminous organ of the squid,Loligo duaauceli, and cuttlefish, Sepia sp.

Delianis Pringgenies, Soelaksono Sastrodihardjo, Noorsalam RahmanNganro & I Nyoman Aryantha

Pringgenies,D., S.Sastrodihardjo, N.R.Nganro & I.N.Aryantha. 2001. Bacterialsymbionts in the luminous organ of the squid, Loligo duaauceli, and cuttlefish.Sepia sp. - Phuket Marine Biological Center Special Publication 25(1): 1.45-1.48.

The luminous organs of the squid L. duaauceli and cuttlefish Sepiasp. are locatedon the ventral surface of the ink sac near of the anus. The organs consist of a lens,the luminous sac, reflector tissue, and the ink layer covering the reflector. Thebacteria, Photobacterium phosphoreum, appear in pockets of the luminous sac.

Bacteria from Loligo duaauceli have a bright blue luminescence, while bacteriafrom the cuttlefish Sepia sp. have a yellow luminescence.Symbiont bacteria only were found in squid after they had hatched, indicatingthat they came from free living bacteria in the water. They were not transferredvia the egg.

Delianis Pringgenies. Marine Science Dept, Diponegoro Uniaersity - Semnrang,Indonesia.

Soelaksono Sastrodihardjo, Noorsalam Rshman Nganro I I Nyoman ArynnthaLife S cience D ept, Institute Te chnolo gy B andun g - B andung, Indonesia.

E - m ail D eli snis P r in g g eni e s : p r in g ge ni e s@y aho o. c o m

INTRODUCTIONFor unknown reasons, emisssion of light byorganisms is far more prevalent in the marineenvironment than on land or in fresh water(Hasting & Morin 1991). Light organs havebeen described in euphausids, shrimp, fishand squid. About 47 %fromtotal of 1'39 generaof squid have light organs (Hasting & Morin1991). The genus Loligo harbours symbiotic,luminous bacteria in paired organs lyingagainst the ink sac near the anus (Herring 1977;

Pringgenies & Jorgensen 1994). The use ofbacterial symbionts as a source of light seems

to be restricted to cephalopods and fishes.Nesis (7982) pointed out that two different

types of luminencence in cephalopods hasbeen demonstrated: (1)by means of symbioticbacteria Photobacterium located in glands ofthe ink sac. (2) the other type is called intrinsicluminescence, which is caused by the

oxidation of luciferase in specialized cells. Inthis study we investigate the transfer ofbacteria to the light organ of juvenile squidand cuttlefish.

MATERIALS AND METHODSCollection of squid were carried out in JeparaBay (Central Java) and Situbondo (East java)at a depth of about 30 m. After removal fromthe net, the squids were put in an ice box tokeep the bacteria alive. In the laboratory of theBrackish Water Culture/BBAP-Iepara, thelight organ was removed and bacteria isolatedon marine agar (MA). The bacteria wereidentified according to Cowan (1.974),Baumann et al. (7984), and Holt et al. (1994).

Fixation nnd embedding. - For histology themantle and ink sac were removed andimmerse d in 3% glutaraldehyde / formalde-hyde in 0.1 M phosphate buffer. Fixation and

I46 Tropical Marine Mollttsc Programme (TMMP)

embedding were done at Aarhus University,Denmark (Pringgenies & Jorgensen,Igg4) andat Airlangga University, Surabaya, Indonesia.The whole light organs was embedded forinvestigation under the light microscope,while each light organ was divided into fourparts before being embedded for examinationunder the electron microscope.

After a short rinse the tissue parts wereimmersed in 1 % osmic tetroxide in 0.1 Mphosphate buffer for 30-60 minute. Followinga rinse in buffer the ink sacs with luminousorgans were transferredtoT0% ethanol. Afterat least 24 hours the tissue was furtherdissected and dehydrated in ethyl alcohol ofprogressively increasing strength. Finally theorgan was embedded in Araldite and placedin an oven at 40 oC over night. The next day itwas moved to 60 'C over night. Series of 2 mmsections were examined under the lightmicroscope. The sections were cut with glassknives on a Jung microtome and stained withtoluidine blue. For electron microscopy, ultra-thin sections were cut with a diamond knifeon a LKB ultramicrotome. Thickness rangedbetween 0.006 and 0.01 mm. The sections werecontrasted with uranyl acetate for 20 minuteand lead citrate for B minutes at roomtemperature and examined under a PhillipsCM 20 electron microscope.

RESULTSLight organs are located on the ventral surfaceof the ink sac near the anus. The size of theink sac is 2-2.5 cm. The light organ measuresapproximately 5 mm in diameter. The lightorgan is easily located from the position of itstransparent lens. Sections studied in the lightmicroscope show that the luminous organconsists of reflectoq, the luminous sac, a lensand an ink layer covering the reflector(Fig. 1). Bacteria appear as granules concen-trated in small pockets inside the luminoussac. Bacteria can also be found on the outsideof the luminous sac. The luminous sacappeared to be divided into many pocket-likechambers. Some of the pockets contained verydense clusters of bacteria. The ink layer was

thick with ink granules covering the luminoussac from the reflector to the space between thereflector and the lens. This layer extendedthrough the inside of the ink sac. Individualbacteria were round, or oval-shaped whenseen in sections. They have a diameter of 0.31-0.92 mm and the length varies from 0.52-3.2mm.

Bacteria cultured on marine agar (MA)wereluminescent; from Loligo duauuceli with a

bright blue luminescense, from Sepin sp. with

Figure 1. Transversal2 7zm light microscopicallvsections of the luminous organ of squid. (a) :cupshaped reflector covered by the luminous sac.(b) : luminous sac containing a large number ofbacteria separated in numerous chambers. (c) : lenssituated on the surface of the ink sac. (d) : ink lavercovering the luminous sac from the reflector to thespace between the reflector and lens. This laverextends through the inside of the ink sac.

Figure 2. Section of the luminous sac with bacteriacontaining poiy-b-hydroxybutyrate (PHB) (a). E\{30,000 x.

a yellow luminescence. We found that thebacteria belonged to the genus Photobacteriunt.Under the electron microscope the bacteriacontained granules of poly-b-hydroxybutyrate

s{Fs

(PHB) seen as white areas surrounded by a

non-unit membrane ( Brock & Madiganl99l)(Fig. 2). Further evidence for the identity ofPhotobqcterium was that they would not growon thiosulphate citrate bile salt sucrose agar(TCBSA) They were gram negative, sensitiveto Z, -diamino 6,7 disopropyl pteridine, andwithout flagellae. Other characters ofPhotobocterium phosphoreum were that theytested positive with 2-3 butanidiol, while theytested negative with gelatine, indole, sucroseand lactose.

DISCUSSIONThe details of photophore structure may varygreatly (Nesis, 1982). For example, Jorgensen& Munk (7979) pointed out that the serialphotophores of the fish Thysanactis dentes aresurrounded by a thin pigment mantle exceptventrally, where a layer of flattened cellsseparated it from the surface of the body. Theelectron micrographs failed to show anyevidence of crystalline or other reflectiveelement in this layer, nor was any reflectionseen by incident illumination of lightmicroscopic section. Munk & Bertelsen (1980)

reported that the luminous gland of angler fish(Chaenophryne draco) has a central lumensurrounded by an epithelium organized as

branched tubular gland. The gland is almostcompletely enclosed within an inner reflectingand an outer pigmented layer.

In the squid Loligo duaauceli and cuttlefishSepia sp. there are a large number of pocketsin each luminous sac. The bioluminescence isassumed to be produced by round- or ovalshaped bacteria harboured within the capsuleof the luminous sac. The bacteria densitydiffers from pocket to pocket. The size of thebacteria in those pockets is quite uniform.

Pierantoni (1914, 1918) and Meissener (1926)

pointed out that the bacterial symbiontspassed from generation to generation troughthe egg. On the other hand, Mortara(1924) andKishitani (1928) pointed out that newgeneration of hosts acquire their symbiontfrom a free-living population in theenvironment. Our electron microscopy study

S maII er Re s e ar ch C ontr ib utions 147

of the early stage development of the squidLoligo dua auceli showed that symbiont bacteriawere found only in the newly hatched samplesnot in the eggs.

Bacteria are also present in the cephalopodsSepiola atlqnticu, Spirula spirula (Herring et nl.1,98I), and angler frsh Chaenophryne draco(Munk and Bertelsen, 1930). Flowever, theywere located in different organs of theseaquatic animals. Bacteria appear in pockets ofthe luminous sac of Sepiola atlqntica andLoligodua uuceli. In Spir ula sp ir ula and Chaenophry ne

draco, they occur in luminous sacs.

Species of symbiotic bacteria in the lightorgans of squid differ according to the species.Nesis (1982) pointed out that the bacteria inlight organs of marine organismbelong to thegenera Vib r i o and P ho t ob a c t er ium. ttuby (199 6)identified symbiotic bacteria of Euprymnamorsei, E. scolopes and E. tnsmanicn tobe Vibrio

fischery. Herring et nl. (795I) found thatbacterial symbionts of Sepia sp., Sepiola ntlanticaand Sepiola robustq were Photobacterium fischery .

We found that bacterial symbionts of Loligoduaauceli and Sepia sp. were Photobacteriumphosphoreum.

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