〔水産増殖45巻2号231-239SUISANZOSHOKU(1997-H9)〕

Filamentous Bacterium, 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℃. The colonies pre-

sented distinctive fingerprint-like features (Fig.2).

This bacterium developed long filaments attached one

end near the broth-air interface in 48 h after inocula-

tion. Under microscopic observation, the filament con-

sisted of short cylindrical cells,2-4×2-3,rim in size.

The filament was about 2-4μm wide and varied in

length up to 12 mm (Fig.3). One end of the filamet

attached the substrate by holdfast (Fig.4), another

end was free or formed bead-like structures and disar-

ticulated into gonidia which were capable of gliding on

substrate,2-3×2-8μm (Fig.3). Gonidia adhered

together forming rosettes (Fig.5). Young rosettes Leucothrix mucor from phyllosoma of spiny lobster 235

had the abilityto glide.This isolatereduced nitrateto 6.0-8.0% NaCl and freshwater. The optimum salinity nitriteand degraded gelatin,but itcould not hydrolyze for growth of this isolate was 1.5%synthetic seawa- casein or starch.It had no abilityto ferment citrateas ter, and 1.0% NaCl. asole carbon or to degrade tryptophan. This isolate did not deposit sulfur granules in cells even in the pH rangefor growth high-sulfurcondition. Observation of pH range for growth of the NJM 9499 The isolateutilized a variety of carbohydrates and showed that bacterialgrowth proliferatedbetween pH derivatives.Simple sugars which supported growth 5.8 until pH 9.5, but no growth appeared at pH 4.6 were monosaccharides such as glucose,fructose, man- and 5.2(Fig.9).The optimum pH for growth of this nose, but not xylose and rhamnose;disaccharides in- isolatewas 7.6. chided maltose, and cellobiose,but not lactose and

trehalose.This isolatewas unable to utilizetrisacchar- Temperature range for growth

ides such as starch,and sugar alcoholsuch as mannitol. As shown in Fig.10, the isolate NJM 9499 could

Glutamine and cysteinewere the amino acids which grow slightly at the low temperature range of 5-15

this isolatewas unable to utilizeas combined carbon ℃ and grew better at the 20-35℃ range. The tempera-

and nitrogensources, whereas lysine,asparagine, argi- ture for good growth was 30℃. The isolate could not

nine and ornithine were suitable organic nitrogen grow at 5℃.

sources.

Artificial infection test

Salinity range for growt The results are shown in Table 2. Mortality of the

hAs the resultsof the experiment in Fig.8, the iso- control group was 6.7%. The group challenged with

late NJM 9499 could apparently grow in sterilized 3.2×105 cells/ml of gonidia had 33.6%mortality,

syntheticseawater at the range of 0.3%to 6.0%, and while the group challenged with 1.6×105 cells/ml of

remained alive in 7.0%synthetic seawater. In NaC gonidia had 20.0%mortality. Phyllosoma in both chal-

l solution,apparent growth was shown between 0.3% to lenged group, demonstrated the filaments of the isolate

4.0%.This strain remained viabilityin 5.0% NaCl. NJM 9499 on the body surfaces and appendages , low

No growth appeared in 8.0% synthetic seawater, locomotive ability and sinking down to the bottom.

Fig.8. Comparison between various concentrations of synthetic seawater and NaCl on growth of L. mucor NJM 9499. 236 N.Kitancharoen et al.(1997)

Fig.9. Effects of various pH on growth of L. mucor NJM 9499.

Fig.10. Effects of various temperatures on growth of L. mucor NJM 9499.

Antibiotic sensitivitytest Table 2. Mortalities(%)of phyllosoma of spiny lobs- ter after challenge with gonidia of L. mucor hi vitro MIC of six antibiotics for the isolate in OZR NJM 9499 broth incubated at 25℃ for 48 h are shown in Table

3.This isolate was effectively inhibited by ampicillin

and gentamycin sulfate(3.1μg/ml).Streptomycin sul-

fate and kanamycin monosulfate were also effective to

the isolate at 6.25μg/ml and 25μg/ml, respectively,

whereas oxytetracycline inhibited this isolate at 100

μ g/ml. Leucothrix mucor from phyllosoma of spiny lobster 237

Table 3. Minimum inhibitory concentrations of anti- to utilize rhamnose, xylose, and trehalose as sole car- biotics against L. mucor NJM 9499 bon sources. Most strains of Thiothyix, a closely re- lated genus, could not utilize sugars such as glucose, fructose, mannose, etc.20).In the case of amino acids, the isolate could use lysine and asparagine, but not glutamine and cysteine, contrary to the strain isolated from wastewater22). This Leucothrix sp. NJM 9499 could use nitrate as a nitrogen source, similar to many , a closely Discussion related genus23).

Leucothrix mucor NJM 9499 isolated from phyllosoma The isolate reported herein could grow at a wide of spiny lobster exhibited peculiar fingerprint-like col- range of salinity,from 0.3%to 7.0%in synthetic sea- onies on OZR agar, and multicellular filaments in OZR water, the same as marine strains reported by Kelly broth. Knot formation, one of the unique morphological and Brock24), but the optimum salinity of the isolate features, as reported by Snellen and Raj19), was not was 1.5%in synthetic seawater, lower than those iso- present among slide culture studies. Gliding gonidia lates. The isolate required NaCl for growth with a and young rosettes could be found through observa- range of 0.3%to 4.0%, the optimum being 1.0%, tion. The rosettes increased in length for 96 h and then which differs from the neotype of L. mucor Oersted stopped, probably because of oxygen and/or nutrient 1844 established by Brock25)in which the maximum is degradation. Gonidial formation increased when the 7.0% NaCl and the optimum is 1.5% NaCl. The opti-

rate of growth was slowed down by the effects of con- mum pH and temperature of this isolate was 7.6, simi- ditions such as anaerobic situation, nutrient deficiency lar to the optimum pH and temperature of Leucothrix

。r temperature reduction2,4). Harold and Stanier14) strains in the laboratory reported by Kelly and observed that rosette formation depends on the condi- Brock24). tion of substrates and the distance between gonidial As shown by the results, the isolatecaused low mor- cells. Holdfasts were found in this isolate. In contrast, tality in healthy phyllosoma. However, its entangle-

Beggiatoa, a closely related genus has no holdfast and ment on the appendages and setae of phyllosoma dis-

rosette formation features20). Bulbous cells generally turbed their locomotive ability and made them sink

appeared without knot formation. This may have been down to the bottom. In the case of weakened larvae, it caused by the enlargement of cells or the fusion of might be the coordinating factor that induced high

adjacent cells as described by Raj2).The GC content of mortality. Although Leucothrix has not been reported

L.mucoy was from 46.9-51.0mole% by byoyant densi- as pathogenicbacterium 1,26), it may be involvedin ty determination20), little higher than this is・late, high mortalityby interferingin the buoyancy of eggs which might caused by different method. The isolate, or respiratorysystem of larvaewhen itbecomes abun-

similar to others in Leucothrix, was catalase positive. dant,especially in moltingstages or in low O226).The

This is one characteristic in which it differs from mucosalsurfaces of eggs and larvalstages support as

Beggiatoa20). Also, Beggiatoa cannot be grown under substratesfor the adhesionof bacteria11).Couch27) the condition of normal oxygen tension2).Studies of suggestedthat the mucoid substanceof the holdfast

Leucothrix nutrition reveal that mannose, peptone, and might block gas diffusion.However, Harper and glutamate are especially good carbon sources, and that Talbot28)couldnot find the significantly relationship xylose is a poor one4). This isolate could utilize fruc- betweenLeucothrix infestation and the lossof lobsters tose, galactose and maltose, contrary to Leucothrix sp. (Homarusspp.).All antibiotics examined in thisstudy

reported by Calomiris et al.21)which could not utilize revealedeffects to controlthe isolate:ampicillin,gen- these sugars. The isolate differs from the wastewater tamycinsulfate and streptomycinsulfate, in particular, strain reported by Williams and Unz22), which is able were effectiveagainst the isolateat lower than 10 238 N.Kitancharoen etal.(1997)

μg/ml.Johnson et al.3)Postulated that 0.1,ug/mlpeni- isms on reared larvae of the European lobster

cillin,5.0μg/ml streptomycin 0.5-0.7μg/ml penicil- Hosyiaritis ga ynfnarus. Fauna 40, 16-19.

lin and streptomycin (1:1 w/w) and 0.7-0.9μg/m 10) Solangi, M. A., R. M. Overstreet and A. L. Gannam. l chloromycetin were effective to L. mucor. The chelating (1979): A filamentous bacterium on the brine

algicide, Cutrine-Plus was also suggested for use to shrimp and its control. Gitilf Research Reports 6,

control L.mucor at 0.1 ppm Cu for 4-6 hr in static 275-282.

treatment, and 0.5 ppm for 24 hr in flow-through 11) Hansen, G. H. and J. A. Olafsen.(1989): Bacterial

treatment 29). colonization of cod (Gadiis fnorhitia L.) and halibut

(Hippoglossus hippoglossitis) eggs in marine Acknowledgments aquaculture. Appl. Environ. Microbiol. 55, 1435-

We wish to thank the Japan Sea Farming Associa- 1446.

tion, Minami Izu Station, who brought the problem of 12) Anderson, J.I.W. and D. A. Conroy.(1968): The

spiny lobster phyllosoma loss to our attention and sup- significance of disease in preliminary attempts to

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イ セ エ ビ の フ ィ ロ ゾ ー マ 幼 生 か ら分 離 さ れ た 糸 状 菌, Leucothrix mucor

Nilubol Kitancharoen・ 畑 井 喜 司 雄 ・原 稔 生

(日本 獣 医 畜 産 大 学,獣 医 畜 産 学 部)

イ セ エ ビ(Panulirus japonicas)の フ ィ ロ ゾ ー マ 幼 生 は 多 細 胞 で グ ラ ム 陰 性 の 糸 状 菌 の 感 染 を 受 け や

す く,こ の こ と で 飼 育 幼 生 が 死 滅 す る こ と が 少 な く な い 。 こ の 度,本 幼 生 の 糸 状 菌 症 を 検 査 す る 機 会 を

得 た こ と か ら,細 菌 の 分 離 培 養 を 試 み る と 共 に,同 定 お よ び 若 干 の 生 物 学 的 性 状 を 試 験 し た 。 分 離 株 は カ タ ラ ー ゼ 産 生,メ チ ー ル レ ッ ド,ゼ ラ チ ン 液 化,糖 分 解(ブ ト ウ糖 ・ フ ラ ク トー ス ・マ ン ニ ッ ト ・マ

ル トー ス ・マ ン ノ ー ス),ア ミ ノ 酸 利 用(リ ジ ン ・ア ス パ ラ ギ ン酸 ・ア ル ギ ニ ン ・オ ル ニ チ ン)が 陽 性

で あ っ た 。 本 株 のGC含 量 は43.7mole%で あ っ た 。 ま た,本 株 は 発 育 至 適 塩 分 濃 度 が1.0%,pHが7.6,

温 度 が30℃ で あ っ た 。 薬 剤 感 受 性 試 験 の 結 果,本 株 は ア ン ピ シ リ ン と ゲ ン タ マ イ シ ン に 強 い 感 受 性 を 示

し た 。 フ ィ ロ ゾ ー マ 幼 生 に 対 す る 感 染 試 験 の 結 果,死 亡 率 は33.6%で あ っ た 。 上 述 の 各 性 状 か ら 本 株 は Leucothri xmucorに 同 定 さ れ た 。