Table Of ContentReference: Biol. Bull. 182: 105-108. (February, 1992)
Embryos of Homarus americanus are Protected
by Epibiotic Bacteria
M. SOFIA GIL-TURNES AND WILLIAM FENICAL
Scripps Institution oj Oceanography, University ofCalifornia, San Diego,
La Mia. California 92093-0236
Abstract. Embryos ofthe American lobster, Homarus attack. This situation is highly analogous to that recently
americanus, are remarkably resistant to infection by the observed for the estuarine shrimp Palaemon macrodac-
fungusLagenidium callinectes, a pathogen ofmany crus- tylus (Gil-Turnes el al., 1989). Embryos ofP. macwdac-
taceans. The surfaces ofhealthy lobsterembryos are cov- tylus were also impervious to attack by L. callinectes. In
ered almost exclusively by a single, Gram-negative bac- thatstudy, Palaemonembryoswere found to hostanepi-
terium, whichgrowsinadense mosaic pattern. In culture, biotic bacterium that produced 2,3-indolenedione, a
this bacterium producesacompound that completely in- molecule toxictoL. callinectes. The intentofthisresearch
hibits the growth ofthe pathogenic fungus in vitro at 10 was to compare Homarus americanus with Palaemon
meg/ml. Large-scale fermentation, extraction, and sub- macrodactylus, and todeterminewhetherlobsterembryos
sequent chromatographic purification led to the identi- are also protected by an association with symbiotic bac-
fication oftheantifungal substanceas4-hydroxyphenethyl teria.
alcohol (tyrosol), an antibiotic substance known to be
produced by terrestrial fungi. Materials and Methods
Introduction Embryo infection experiment with Lagenidium
callinectes
Like several other decapod crustaceans, the American
lobster Homarus americanus incubates its embryos ex- Gravid female Homarus americanus were collected in
ternally, and each femalecarriesa largeclustercomprising the vicinity of Martha's Vineyard, Massachusetts, air
up to 60,000 embryos. The embryos are attached to spe- shipped to California, and maintained in aquaria at the
cialized abdominal pleopods until hatching some nine Bodega Marine Laboratory in Bodega Bay, California
months after fertilization (Cobb and Wang, 1985). (UC-Davis). Theembryoswereobservedtobeatdifferent
Throughout this long brooding period, during which the stagesofdevelopment. Tengroupsoffiveembryoclusters
female is said to be "in berry," the embryos are contin- each were detached from the females. Each cluster was
uously exposed to water-borne microorganisms. It is re- rinsed with three aliquots ofsterilized seawater and sub-
markable that the seemingly unprotected embryos can sequently suspended from acotton thread in aerated 125
survive microbial encroachment. The phycomycetous ml Erlenmeyerflaskscontaining 75 mlofsterileseawater.
fungus Lagenidium callinectes is a pathogen of many A liquid suspension ofLagenidium callinectes was pre-
crustaceans. Larvaeandjuvenilesofthe American lobster, pared by homogenizing a 0.5 cm diameter agar core of
when kept in unnatural conditions, e.g.. in aquaria, are fungal hyphae in 10 ml of2216 Difco Marine Broth me-
extremely vulnerable to infection by this fungus (Fisher dium. The fungusgrew foroneweek, andtheculture was
el al, 1976; Nilson el ai, 1976; Provenzano, 1985). In then shaken vigorously to break up the hyphae. Aliquots
contrast, Homarus embryos, even when detached from of 1 ml ofthis thick suspension were added to each ex-
the female, appear to be remarkably resistant to fungal perimental flask. Addition offungal culture was repeated
after the fifth day on a daily basis for a period of two
Received 31 July 1991;accepted 7 October 1991. weeks.
105
106 M. S. GIL-TURNES AND W. FENICAL
Isolation andculture ofassociated bacteria Pure bacterial films were prepared as follows: a drop ofa
Inatypical experiment, fiveembryos from each animal 3-day liquid culture was deposited on a small millipore
were homogenized in an autoclaved tissue grinder with filter (0.25 n pore size) placed on an agar plate. As soon
10 ml ofsterile senvater. One drop ofthe homogenate, as growth was visible, the specimens were fiMxed in 3%
sa2u2nb1dc6uolfMtau1rr/ei1dn0eaaftnAedgra1ri-/31pl0wa0eteedsk.isl.uCtoAilloontnsh,ioeuwsgehrweetrhpreleaertoeerdmofoovnuerdDmiaofnrc-do wcfaaocrsomhdaeyldldatethheryedetretiihamyneddsraft3oer%5bgumflfiuetnrair(napl0dH.e2h7My.d4e)caifcnoord0y.1l2ahteasnosdloudttiihouenmn.
phologicallyvariablecoloniesweregenerallyobserved,one The specimensMwere then transferred to 2% osmium te-
distinct bacterium (SGT-76), a salmon-colored, slow- troxide in 0.2 cacodylate solution for 1 Mh, and subse-
growing (at 21C), Gram-negative rod, was consistently quently rinsed six times for 5 min in 0.2 cacodylate
obtained. In liquid culture, thisstrain inhibited thegrowth solution. After dehydration using a sequence ofethanol/
otifnLg.acgaallrinceocrleess,. A0n.5ticfumngianldteisatmientgewr,asfpreormfolramwendsboyfctuhte- dwiesrteilclreidtwicaatle-rpotirnetadtrmieendtusnfdoerr1C0Om2i,ncoeaatche,dtwhiethspgoelcdim(e3n0s0
pure bacterium and placing them on agar plates approx- AS)4,50aAndSEmMi.crographs obtained with a Hitachi Model
imately 1 cm from agarcorescontaining radiating hyphae
ofL. callinectes. Because ofthe inhibition observed, this
bacterium was selected for subsequent chemical studies. Results
After 18 days and ten additions of fungal culture, H.
Extraction andpurification ofthe antifungalcompound americanusembryos appeared healthy and free offungal
The Gram-negative, salmon-colored bacterium, SGT- infection. The visible organ anatomies and heartbeat rates
76,wascultured, at21 C, ina 16-1carboyusingamedium ofthe treated embryoswere identical to those ofthecon-
composed of3 g BactoPeptone (Difco) and 5 g yeast ex- trols. Scanningelectron micrographsshowedthattheem-
tractperliterofseawater. Theculturegrew, with aeration, bryonal surface was covered by an almost monoculture
forthreeweeks. Thefinal pH ofthe medium was8.8. The of a rod-shaped bacterial strain (Fig. 1A). Some of the
entire culture was extracted twice with 4 1 ethyl acetate. embryos, at different stages ofdevelopment, were occa-
Afterevaporation ofthecombinedsolvents, theremaining sionally found to have verysparsecoverage bythree mor-
crude extract was fractionated by silica-gel vacuum flash phologicallydifferentbacteria(Fig. IB), inadditiontothe
chromatography using variable amounts ofethyl acetate rod-shaped strain. Older embryos, near hatching, were
in isooctane. The antifungal activity ofeach fraction was consistently observed to possess dense coverage by an al-
determined by placing 0.5 mg ofeach dry fraction onto most monoculture ofthe rod-shapedbacterium (Fig. 1C).
a 0.5 cm paper disk and placing the disk at the edge of Replicate inoculations of embryo homogenates on
fungal growth. The active compound eluted with 80% MarineAgarplatesresultedintheisolationofa maximum
ethylacetate/isooctane. Finalpurification oftheantifungal offour, but usually fewer, strains ofbacteria. One ofthe
compound was achieved by size exclusion chromatogra- strains(SGT-76), which wasconsistently isolated, was in-
phyon Sephadex LH20 usinga mixture ofhexane/meth- hibitorytoL. callinectes. Thisbacterium, aGram-negative
ylene chloride/methanol (2:5:1). The purified compound rod insensitive to penicillin, was a pale salmon-colored
wascharacterized by infrared spectroscopy (IR), by high- strain, and it was extremely slow growing on agar plates
resolution mass(HRMS)and by 'H and I3Cnuclearmag- and in liquid medium. For reasons unknown, the pH of
netic resonance spectrometry (NMR). the culture medium seemed to rise to 8 or more during
fermentation. This rise in pH could provide a possible
Scanning electron microscopy explanation forthe poorgrowth observed. Scanningelec-
tron micrographs ofthis bacterium, grown on millipore
Embryoswere fixedin 2.5%glutaraldehydein 3% saline filters, showed that the cells were identical in size and
solution fora linimum of24 h. Afterthree rinses in 3% shape, and had an identical growth pattern to those ob-
salinesolutio: >in, each specimen wastransferred served on the surface ofthe natural embryos (Fig. ID).
toasolutionof i iiumtetroxidein 3% salinesolution An antifungal compound produced by the bacterial
for nine minutes. were then stored in saline straingrown in liquid medium wasisolatedandidentified
solution overnight .equentlydehydrated usingan astyrosol, 4-hydroxyphenethyl alcohol (Fig. 2). Theactive
acetone/distilled water ; ic-nce: 35% for 15 min, 50% compound wasisolatedasa viscousoil which showedthe
for 15 min, 75%for30min,95%for 1 h,and 100%ethanol following spectral characteristics: IR (film): 3400, 3150
for 12 h. Critical point drying was done under CO2 and cm"1; HRMS requires 138.04 forC8H, O2, found 136.06;
the gold coating thickness was 300 A. Electron micro- 'H NMR, 200 MHz (acetone-do): 7.0 (d, 2 H, J = 8.6
graphs were obtained with a Hitachi Model 539 SEM. Hz), 6.6 (d, 2 H, J = 8.6 Hz), 3.7 (t, 2 H), 2.7 (t, 2 H);
H. AMERICAM'S EMBRYOS PROTECTED BY BACTERIA 107
\
Figure 1. Scanning electron micrographs ofhealthy embryos ofHomarus americanus under various
conditions. (A)SurfaceofembryoafterexposuretothefungusLagenidium callinectes. illustratingthelack
offungalattachment(1250X). (B)Surfaceofembryoshowingthecoveragebythecolonial rodmorphotype
andtheotherthreetypesoccasionallyfound(1000.' ). (C)Surfaceofembryo,atnearfullgestation,showing
extensive and thick coverage by the rod-type bacterium (5000X). (D) Micrograph ofthe pure bacterium
grown ona millipore filter(2000X).
I3C NMR, 200 MHz (acetone-d6): 155.0, 131.0, 130.0, particularstrain, thus health and successful development
1 15.5, 1 15.4, 63.8, 39.1 (Fig. 2). Allchemicalandspectral appear to be related to the degree ofbacterial coverage.
datawere identical to those from thecommercially-avail- Harper and Talbot (1984), who investigated embryos of
able 4-hydroxyphenethyl alcohol (tyrosol, Aldrich several Homarus species to determine ifthe presence of
#18,825-5). Tyrosol effectively inhibitedgrowth ofL. cal- epibiotic bacterial flora was related to loss of embryos
linectes in liquid culture at a concentration of 10 meg/ from the pleopods, also observed four bacterial morpho-
ml. In agarplate assays, 100 megtyrosol perdisk resulted types. Their bacteria appear to be morphologically iden-
mm
in an 8 zone offungal inhibition. tical to those described in this article, including one that
they described as a "colonial rod." They found that em-
Discussion bryos from wild born and wild spawned H. americanus
wereheavilycovered bybacterial rods, andthattheseem-
All the embryos observed were covered largely by a bryoswere successfully retained by the adult until hatch-
single, rod-shaped bacterium, distinguishable from other ing. Based upon the repetitive isolation of the salmon-
types by its characteristic dense, mosaic-like growth pat- colored bacterium (SGT-76) from healthy embryos, and
tern. Theoldestembryoshadthethickestcoveragebythis its highly characteristic mosaicgrowth pattern on natural
108 M. S. GIL-TURNES AND W. FENICAL
Studiesofthebacterial symbiontsofcommercially im-
HO portant marine invertebratescouldprovide important in-
formation leading to the control ofdisease under aqua-
OH
culture conditions. Indeed, there is a significant need to
develop inexpensive and environmentally safeantifungal
agents forthis specific application. The simple molecules
discussed here, which appeartobederived from thecom-
mon amino acids tyrosine and tryptophane, should be
4-hydroxyphenethyl alcohol considered in this application.
Acknowledgments
"tjrosol"
Figure2. Chemicalstructureoftheantifungalmetabolite4-hydrox- We would like to thank Ashley Yudin, William S.
yphenethyl alcohol. Fisher, Wallis H. Clark, and Prudence Talbot for their
assistance in this study. The staffand students at the Bo-
dega Marine Laboratory wereextremely helpful and their
surfaces and on filters, we believe that this bacterium is cooperation is gratefully acknowledged. The work is
the natural epibiont of Homams embryos. At the same mainly a result ofresearch sponsored by NOAA, National
time, we recognize that this proposal will be difficult to Sea Grant College Program, Department ofCommerce,
rigorously prove. undergrant #NA85AA-D-SG140, project number R/MP-
The antifungal compound produced by bacterium 39, through the California Sea Grant Program, and in
SGT-76, 4-hydroxyphenethyl alcohol, ortyrosol. haspre- part by the California State Resources Agency. Portions
viouslybeenreportedasanaturalproduct fromtwofungal ofthis research were also supported by the National In-
species that are involved in symbiotic associations with stitutes ofHealth, NCI, undergrant CA44848. The U. S.
plants (Stoessl, 1969; Claydon et til., 1985). In an appar- Government isauthorized toreproduceanddistributefor
entlysimilaradaptation, those fungi seem to protecttheir governmental purposes.
hosts against invasion by pathogenic fungi. Literature Cited
Protection ofembryos by epibiotic bacteria has been
shown previously (Fisher, 1983; Gil-Turnes et a!., 1989) Clayadnodn,fNe.e,diJn.gF.deGtreorvreen,tasndfrMo.m PPohpolem.op1s9i8s5.oblEolnmga.barPkhybteoetclheembiosrtirnyg
forthecaridean shrimp Palaemon macmdactylus, alsoan 24(5):937-943.
external brooder. In the present study, the association of Cobb,J.S.,andD.Wang. 1985. Fisheriesbiologyoflobstersandcray-
H. arnericcmusembryoswithaGram-negative, rod-shaped fishes. Pp. 168-248 in TheBiologyofCrustacea. Vol. 10,Academic
bacterium suggests a similar adaptation in which a vul- Press. New York.
nerable host is protected against pathogenic microorgan- Fishweirt,h\aV.quSa.ti1c98b3ac.teriEag.gsBioolf.PBaullal.em1o6n4:m2a0c1m-d2a1c3t.ylus: II. Association
isms by symbiotic bacteria. At least one explanation for Fisher, W. S., E. H. Nilson, L. F. N. Follett, and R. A. Shleser. 1976.
the resistance ofHomams embryos is the bacterial pro- Hatchingandrearinglobsterlarvae(Homamsamericamis)inadisease
duction of the antifungal compound tyrosol in nature. situation. Aquaculture1: 75-80.
Although tyrosol is only a moderately potent antifungal Gil-Turnes,M.S.,M.E.Hay,and\V.Fenical. 1989. Symbioticmarine
bacteria chemically defend crustacean embryos from a pathogenic
agent, thedense bacterial coverage observed would easily fungus. Science246: 117-118.
result in high levels ofthe compound at the embryo sur- Harper, R. E., and P. Talbot. 1984. Analysisoftheepibiotic bacteria
face. Thus, tyrosol could function effectively to reduce oflobster(Homams)eggsandtheirinfluenceonthelossofeggsfrom
fungal encroachment. thepleopods.Aquaculture36: 9-26.
The antifungal agents isolated from crustacean-asso- Nilsoofn,larEv.alH.l,obWst.erS(.HFoismhaerm,saanmderRi.caAm.isS)h.leJs.erIn.ve1r9l7e6b.r. PAathnoel.w2m7y:c1o7s7i-s
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a Palaemon macrodactylus-associaled bacterium), are Provenzano,A.J.,Jr. 1985. Cultureofcrustaceans:generalprincipals.
simple moleculeswithonly modest potencies. Thesemol- Pp.279-280in TheBiologyofCrustacea, Vol. 10.EconomicAspects:
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Stoessl, A. 1969. 8-Hydroxy-6-methoxy-3-methylisocoumarin and
dium, however, perhaps suggesting that they are targeted othermetabolitesofCeratocyslisfimbriata. Biochem. Biophys. Res.
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