〔水産増殖45巻2号231-239SUISANZOSHOKU(1997-H9)〕 Filamentous Bacterium, Leucothrix mucor from Phyllosoma of Spiny Lobster, Panulirus japonicus Nilubol KITANCHAROEN, Kishio HATAI, and Naruo HARA Nippon Veterinary and Animal Science University,1-7-1 Kyonan-cho , Musashino, Tokyo 180, Japan Abstract A multicellular, Gram negative, filamentous bacterium, found in phyllosoma of spiny lobster Panulirus japonicus from Shizuoka Prefecture, Japan , was isolated and identified as Leucothrix mucor Oersted. It was catalase positive and unable to deposit sulfur granules even in sulfur- containing media. It had the ability to utilize some carbohydrates and derivatives , as monosaccharides, disaccharides and sugar alcohol, but not trisaccharides . This isolate could utilize some amino acids. The GC content of the isolate was 43.7mole%. The salinity range for growth was 0.3-7.0% in synthetic seawater and 0.3-4.0%in NaCl, with optima of 1.5%in synthetic seawater and 1.0% in NaCl. The pH range for growth was from 5.8 to 9.5 , with an optimum pH of 7.6. This isolate could grow between 5℃ and 35℃, but grew best at 30℃ . Ampicillin and gentamycin sulfate were the most effective antibiotics against this isolate. The mortality of phyllosoma challenged with gonidial suspensions of 3.2×105 cells/ml and 1.6×105 cells/ml were 33.6% and 20.0%respectively, whereas the control group had 6.7%mortality. Leucothrix is a filamentous, obligatory aerobic, Blomg)reportedthe occurrence L.mucorof onEu marine bacterium,occurring in the intertidalzones of ro-pean lobster,Homarus gammarus.Brine shrimp.Arte- both temperate and tropical regions1,2).It normally mia saliva,typical feed of shrimpsand fishes,also ex- lives as an epiphyte on macroscopic marine algae and hibitedthe overgrowthof a L.mucor-like strainlo) arthroPods3,4),and is also found in decomposing algae Hansen and Olafsen11)reportedthe occurrence of and marine detritus3).However,one strictlyfreshwa- Leucothrixsp., particularly on pre-hatchingeggs of ter strain has been reported from pertrochemicalwas- cod, Gadus morhua L. and halibut,Hippoglossus hippog- tewater by Poffte elal.5).Leucothrix has frequentlybeen losses.Leucothrix sp. was also demonstratedon rock reported to occur in activatedsludges6,7). crab,Cancer irrotus, green crab, Carcinus mamas, grass Eggs and larval stages of cultured marine animals shrimp,Palaemonestes pugio and penaeidshrimps such often appear with the typical filaments of Leucothrix as Penaeidstylirostris, P.setiferus and P.vannamei3). sp., especiallyin cultured lobster.Steenburgen and Anderson and Conroy12)Postulated that the presence Schapiro8)reportedthe infestationof L. mucor Oersted of Leucothrix-likebacterium was relatedto egg mortal- in the American lobster,Homarus americanus juvenile ity of reared English prawn(Paleomon serratus). stages cultured under intensive conditions.Dale and This study was carried out on the filamentousbac- Received :November 1,1996 Key words:Leucothrix mucor, Phyllosoma, Spiny lobster, Filamentous bacterium 232 N.Kitancharoen catal.(1997) terium isolatedfrom diseasedphyllosoma of spiny Biochemical characteristic lobster,Panulirus japonicus(von Siebold)in Shizuoka The isolate NJM 9499 was investigated the following Prefecture,Japan. We could not find the causative biochemical characteristics:gram stain, motility, cata- agentdue to thegradual deaths of phyllosoma,only the lace, citrate, oxidation-fermentation, indole, Methyl filamentousbacterium heavily entangled on the body red, Voges Proskauer, nitrate utilization,gelatin de- surfaceof the moribund phyllosoma.This was re- gradation, casein hydrolysis, carbohydrate utilization, garded as one factorthat might be inducedmortality. amino acid utilization.All amino acids were L-isomers. Morphological,biochemical and physiologicalcharac- Sulfur deposition was also carried out according to teristicsof thebacterium were investigated.Sensitivity Williams and Unz18). of the isolateto antibioticswere also determined. Pathogenicityof the bacteriumagainst phyllosoma of spinylobster by artificialinfection wasSalinity done. range for growth The gonidia of the NJM 9499 was centrifuged at 3700 rpm,15 min. and washed 2 times with sterilized Materials and methods distilled water. Synthetic seawater and NaCl were pre- pared at double concentrations of O.3,0.5,1.0,1.5, Morphology and GC content Phyllosoma of spiny lobster, P.japonicus were 2.0,2.5,3.0,3.5,4.0,5.0,6.0,7.0 and 8.0% final obtained from Shizuoka Prefecture in November,1994. concentrations.1.0ml of the gonidial suspension in The filamentous bacterium was detected under light sterilized distilled water was set in 1.0ml of each salt microscopy (Fig.1). Isolation of the bacterium into solution, and incubated at 25℃ for 48 h. In all experi- culture was accomplished by washing excised parts of menu, the initial suspension of gonidia was also fixed the phyllosoma appendages with sterilized synthetic by a drop of formalin as the initial control suspension, seawater 3 times then drawing onto OZR agar plates and the tubes in which the bacterial growth could not (formula as Sieburthl3)).All plates were incubated at be distinguished by the naked eye, was one loop taken 25℃.The single colony was transferred onto OZR and streaked on OZR agar to examine the viability, fol- agar and maintained for use in all experiments. Subcul- lowing which a drop of formalin was put into each ture was performed at 4 wk intervals. tube to inhibit growth. All tubes were centrifuged and The present isolate, namely NJM 9499, was inocu- washed with sterilized distilled water 2 times, then lated in OZR broth and incubated at 25℃ for 48 h, sonicated 15 sec on ice. Growth was determined by then changed to 5℃ for 48 h to induce gonidia forma- measuring optical density at 600 nm compared with the tion and harvested by filtering through sterilized initial control suspension. Whatman filter no.14). Slide culture as described by Harold and Stanier14)wasperformed to follow the pH range for growth bacterial life cycle. The gonidia of the isolate NJM 9499 were harvested The GC content was determined by high perform- and washed 2 times with sterilized distilled water. ance liquid chromatography (HPLC)using a TOSOH Then 2 ml of the initial concentration suspension was Model CCPD liquid chromatography (Tosoh Co., inoculated into each tube, centrifuged and the upper Japan)fitted with a TSKgel ODS-80Tm column (size portion gradually poured out. OZR broth was adjusted 150×4.6mm i.d., Tosoh Co.).DNA extract according to pH 4.6,5.2,5.8,6.4,7.0,7.6,8.2,8.8 and 9.5,2 to the method of Makino et al.15)was hydrolyzed by ml of each pH media was supplied into the tubes of nuclease Pl(Yamasa Shoyu Co., Japan)and then alka- bacterial patch. The tubes were incubated at 25℃ for line phosphatase according to the method described by 48h, thenceforth growth was measured. Tamaoka and Komagata16). Four equimolar mixtures of nucleotides (Yamasa Shoyu Co.) was used as the standardl7). Temperature range on growth The temperature study was performed by inoculat- ing 0.5ml of gonidia into test tubes containing 1.5ml Leucothrix mucor from phyllosoma of spiny lobster 233 Fig.1. L. mucm NJM 9499 entangled on the body surface of phyllosoma of spiny lobster, Scale bar=50μm . Fig.2. Single colony of L.mucor NJM 9499 on OZR agar after 48 h at 25℃, about 1 mm in size. Note fingerprint- liked characteristic. Fig.3. The typical filamcnts of L. mucor NJM 9499 in OZR broth composed of short cyhndrical cells, Scale bar=50μm. Fig.4. Holdfast organ for attaching substrates. Scale bar=50μm. Fig.5. Gonidia(G),gonidia adhered together forming young rosette(R)after 24 h in slide culture . Scale bar=30μm. Fig.6. Rosette formation after 120 h in slide culture. Scale bar=50μm. Fig.7. Different bulbous cells found in slide culture. Scale bar=50μm. 234 N.Kitancharoen etal.(1997) of OZR broth, and incubated at various temperatures, composed of 3-5 gonidia could be observed after 24 h 5,10,15,20,25,30,35 and 40℃, for 48 h. After- (Fig.6). The rosettes gradually increased in length wards, growth in all tubes was determined. for 96 h, and then the development stopped. Bulb formation was also exhibited. In this case, bulbs Artificial infection test formed by fusion of adjacent cellsor spontaneous en- The phyllosoma of spiny lobster, Panulirus japonicus, largement of a singlecell(Fig.7).Knot formation was were acclimatized to laboratory conditions with not present in thisobservation. post-hatching brine shrimps(Artemia sp.)feeding for GC content of this isolatewas calculatedto 43.7 2 days. Then each of 30 phyllosoma were placed into mole% by high performance liquidchromatography de- 50ml sterilized synthetic seawater in 100 ml beakers termination. with aeration, and challenged with gonidial suspen- As the results,the isolateNJM 9499 isolatewas sions of 3.2×105cells/ml and 1.6×105cells/ml. Mor- identifiedas Leucothrix mucorOersted. tality of each group was checked every day for 7 days, compared with the unchallenged control. Dead speci- Biochemical characteristic stitidies mens were discarded daily and investigated under As shown in Table 1, the isolate NJM 9499 was a light microscopy. Gram negative, catalase positive, non-fermentative bac- terium. It produced acid during aerobic growth. The Antibiotic sensitivity test filaments were non-motile, except for gonidia which The gonidia of NJM 9499 were washed with steril- ized distilled water 2 times and diluted for the initial Table 1. Biochemical characteristics of L. mucor NJM 9499 concentration suspension, then transferred into each solutions of the antibiotics in OZR broth with same volume. The antibiotics were prepared at various final concentrations of 100,50,25,12.5,6.2,3.1,1.6,0.8, 0.4 and 0.2μg/ml of active ingredients, then incu- bated at 25℃ for 48 h. Optical density measurement was performed to determine the growth potential. Results Morp hology acid GC crnit ent The filaments of the isolate NJM 9499 entangled on the body and gill surface of phyllosoma of spiny lobs- ter were isolated and held at 25℃.