bacteria associated gut marine copepods · calanoid copepod, hasprovedto beuseful as an...

Vol. 37, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1979, p. 750-7590099-2240/79/04-0750/10$02.00/0

Bacteria Associated with the Surface and Gut of MarineCopepods

M. R. SOCHARD, D. F. WILSON,t B. AUSTIN, AND R. R. COLWELL*Department ofMicrobiology, University ofMaryland, College Park, Maryland 20742

Received for publication 16 January 1979

Little is known about the nature of bacteria associated with the surface and gutof marine copepods, either in laboratory-reared animals or in the natural environ-ment. Nor is it known whether such animals possess a gut flora. The presentreport deals with studies of microorganisms isolated from healthy, laboratory-reared copepods of the species Acartia tonsa Dana, from several species of wildcopepods collected from a marine or estuarine environment, and from laboratorydishes containing moribund copepods. Evidence for a unique gut flora in labora-tory-reared animals is presented; the predominant bacteria were represented bythe genus Vibrio. Other organisms such as Pseudomonas and Cytophaga werefound less abundantly associated with the copepods and not specifically associatedwith the gut.

Acartia tonsa Dana, an estuarine and neriticcalanoid copepod, has proved to be useful as anexperimental animal in standardized laboratoryculture (25). However, little is known about theeffects of associated microorganisms on suchmarine invertebrates, or, in fact, about the actualassociations themselves, either in the naturalenvironment or in laboratory cultures. Ecologi-cal relationships and changes in such relation-ships that might occur when wild copepods arebrought into culture should be understood, es-pecially if there is a unique association betweencopepods and bacteria, such as symbiosis orcommensalism. Mass mortalities frequently ob-served in laboratory culture of copepods may becaused by specific pathogens or opportunisticbacteria, but little is known about bacterial path-ogens infecting copepods. In addition, the gutflora of the copepod may confer protection onthe animals, but until the existence of a com-mensal gut flora in the copepod is established,such an hypothesis cannot be proven.

Research for the project reported here wasinitiated on a cruise in the Gulf of Mexico aboardthe U.S.N.S. MIZAR. Several species of cope-pods were collected at four different stations,including sites in the open ocean, off the conti-nental shelf, and in the Mississippi Delta. Sam-ples were also collected in the Anclote River,Tampa, Fla.; in Bayboro Harbor, Bayboro, Fla.;and from copepod cultures at the Naval Re-search Laboratory, Office of Naval Research,

t Present address: Marine Biology and Biochemistry,Ocean Sciences Division, Naval Research Laboratory, Officeof Naval Research, Washington, DC 20375.

Washington, D.C. Cultures of bacterial isolateswere preliminarily grouped by selected biochem-ical tests, and strains comprising the predomi-nant flora were subjected to numerical taxon-omy. The relationships noted between bacteriaand copepods and the evidence indicating theexistence of a unique gut flora in A. tonsa Danareared under defined laboratory conditions arereported here.

MATERIALS AND METHODSSample collection. A total of ca. 50 water and 50

copepod samples were collected. Samples were ob-tained at 1 m below the surface at four stations in theGulf of Mexico, located as follows: station 1-Lat.27°00'N, Long. 86°00'W; station 2-Lat. 27°30'N,Long. 88°30'W; station 3-Lat. 29°19'N, Long. 880.30'W; and station 4-Lat. 28°35'N, Long. 89°00'W, inthe Atlantic Ocean. Surface water samples were alsocollected from the mouth of the Anclote River, Tampa,Fla. and off the seawall at Bayboro Harbor, Fla. Ap-proximately 20 water samples were taken from thetanks in which copepods are maintained in continuousculture at the Naval Research Laboratory by methodspreviously documented (10, 25, 26). Approximately 12water and copepod samples from copepod culturedishes showing animal mortality and presence ofslimes and/or ropy filaments attached to dead or dyingcopepods were also included in the analyses. Micro-scopic examinations were made of live and dead co-pepods for epibionts or parasites (i.e., dinoflagellates,fungi, or peritrichous ciliates), but none were observedin the copepods examined.

Processing of samples. Water was collected witha sterile Niskin sampler (General Oceanics, Miami,Fla.). Fifty-milliliter water subsamples were filteredthrough 0.22-,um membrane filters (Millipore Corp.,Bedford, Mass.). Duplicate filters were each placed on

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BACTERIAL ASSOCIATIONS IN MARINE COPEPODS 751

TCBS agar (BBL Microbiology Systems, Cockeysville,Md.), on mycology agar (Difco Laboratories, Inc., De-troit, Mich.) made up in distilled water or 80% sea-

water, and on 2216 agar (Difco). In the field, copepodswere collected by plankton tow, using a 0.5-ni, 250-,im-mesh plankton net during normal trawling operationsat approximately 2 knots. Three copepods of eachspecies were washed twice in 1.0 ml of sterile sea

water, and the second wash was plated on the medialisted above. Bacterial isolates from these specimenswere termed "copepod surface isolates." The copepodswere washed a third time, the wash was discarded, andthe animals were ground in 1.0 ml of sterile seawaterin a tissue grinder. Homogenized copepod tissue was

plated on the media listed above. Bacterial isolatesfrom these samples were termed copepod "gut-sur-face" isolates, since there was no assurance that suchisolates truly represented gut flora alone. It was felt,however, that differences among isolates from gut-surface specimens and those from surface specimensmight indicate which strains most probably were ofgut origin. The question was answered, in part, byexperiments involving aseptic removal of the entiregut from laboratory-reared copepods (see below).

Identification of copepods. Wild copepods were

identified on board ship by members of the MarineScience Institute of the University of South Florida.Copepod species so identified included A. tonsa, Pon-tellopsis regalis, a species of Pleuromamma, Labido-cera aestiva, and Centropages furcatus.Copepod gut dissections. The preliminary work

with washes of copepods showed that aseptic dissec-tion of copepod gut needed to be examined. Thus, gutsamples were prepared by aseptic dissection of labo-ratory-reared A. tonsa. Dissection was performed on

healthy adult copepods, employing sterile technique.Animals were each embedded head first in marineagar 2216. The entire gut was removed by hooking a

fine (3.0-mil; ca. 0.076-mm) sterile tungsten wireembedded in a glass rod into the caudal rami. Therami were dissected away with another sterile wire,leaving the gut entirely free. The gut was maceratedwith a sterile bacteriological loop and plated on TCBSand marine agar 2216 (see below).Media employed for total viable counts. Total

counts of Vibrio-like organisms were made on TCBS.Total viable counts of aerobic, marine, heterotrophicbacteria were made on marine agar 2216. Counts offungi were made on mycology agar (Difco) by employ-ing duplicate plating procedures. One set of plateswere made up with distilled water, and the othercontained 80% filtered seawater and 20% distilled wa-ter. The seawater medium was employed for the pur-pose of isolating fungi requiring seawater for growth.

Cultures were picked from all plates, with onlyrepresentative strains picked from TCBS agar, sincethe purpose of picking colonies from TCBS was toverify the selection for Vibrio by this medium and theselection of colonies from the other media was toestablish the generic distribution of the bacterial floraassociated with the copepod. A total of 329 isolateswere collected, of which 93 were subjected to numeri-cal taxonomy analysis.

Biochemical tests on bacterial isolates. Bacte-

rial isolates were tested for reaction in Hugh andLeifson medium (MOF, Difco) with glucose. Moellermedium (Difco) was used to determine arginine dihy-drolase, ornithine, and lysine decarboxylase reactions.Requirement for sodium chloride for growth was de-termined by inoculating T broth medium consisting of1.0% (wt/vol) Trypticase (Difco), 0.2% (wt/vol) yeastextract (Difco), and none, 3% (wt/vol), 7% (wt/vol), or10% (wt/vol) NaCl added. Kovac's oxidase test (N-discs, BBL) was performed on 18-h cultures grown onmarine agar 2216 (Difco).API 20 strips (Analytab Products, Inc., Plainview,

N.Y.) were used, with a three-salt solution (7) as thediluent. Lecithinase tests were done on marine agar2216, and lipase activity was also determined (7). Chi-tinoclastic activity was assessed by the method ofSkerman (21) on marine agar 2216.

Antibiotic susceptibility tests. Antibiotic sensi-tivity tests were performed using antibiotic disks(BBL) placed on seeded marine agar 2216 plates.Antibiotics tested included tetracycline (Te3o), bacitra-cin (Blo), erythromycin (E15), terramycin (T30), poly-myxin B (PBso), chloramphenicol (C5), novobiocin(NB5), dihydrostreptomycin (DS,o), penicillin (Pio),and aureomycin (A5). The antibiotic vibriostat 0/129(2,4-diamino-6,7-diisopropyl pteridine) was preparedby a modified procedure of Hendrie et al. (11).

Cultural characteristics of bacterial isolates.Bioluminescence was determined by growing the bac-terial strains on marine agar 2216 or on photobacter-ium agar (Difco), followed by inspection in a darkenedroom. Fluorescent and other pigments were detectedon Pseudomonas F or P medium (Difco), preparedwith 80% filter-sterilized seawater and 20% distilledwater, or on marine agar 2216. Characteristics re-corded included turbidity, motility, flagellar stains,temperature requirement for growth, and morphologyas viewed by electron microscopy of negatively stainedpreparations grown in T broth of the following com-position: Trypticase (Difco), 0.3% (wt/vol); yeast ex-tract (Difco), 0.3% (wt/vol); filtered seawater, 75%;distilled water, 25% (pH 7.9). One drop of trace ele-ments solution was added to each 100 ml of the Tmedium. The solution consisted of the following:CuSO4. 5H20, 1.96% (wt/vol); ZnSO4.7H20, 4.4% (wt/vol); CoCl2.6H20, 2.2% (wt/vol); MnCl2.4H20, 36%(wt/vol); Na2MoO4. 2H20, 1.26% (wt/vol); iron seques-trene, 1.0% (wt/vol).Morphology of isolates. The primary isolates

transferred to marine agar 2216 slants were Gramstained. Also, flagellar stains were processed usingstandard methods (14). Electron microscopy, employ-ing negative staining, was also done (4), with thefollowing modification in methodology: potassiumphosphotungstate was prepared in three-salts solutionbrought to pH 7.25 with 1 N KOH to prevent lysis ofthe marine bacteria. Bovine serum albumin was addedto T broth cultures to a concentration of 0.05% (wt/vol). An RCA EMU 2B electron microscope (RCA,Princeton, N.J.) was used for the electron micoscopestudies.

Numerical taxonomy. Preliminary grouping ofthe strains was possible using the scheme shown inFig. 1 and a composite of schema employed by other

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APPL. ENVIRON. MICROBIOL.

BASIC DIAGNOSTIC SCHEME FOR MARINE GRAM-NEGATIVE BACTERIAREQUIRES SALTWATER (W3%) MEDIA

IO/F MEDIUM

I ~~~OXID3ATIVE NO RATOFERMENTATIVE (GAS) FERMENTATIVE (NO GAS) I NO REACTIONOXIDASE + (NO pH CHANGE OR ALKALINE)n-i-i OXIDASE +OR-

OXIDASE- OX10DASE + FLUORESCENT NO FLUORESCENT HIGHLY PIGMENTEDOXIDASE OXIDASE +PIGMENT PIGMENT

ON MEDIUM F ON MEDIUM FI IAS |.0i GLIDING FLAGELLAR MOTILITY

ENTEROBACTERIACEAE AOMOAS PSEUOOAS I MOTILITY OR NON-MOTILEFLLORESCENS PSEU"OVAS

GROUP

CYrOPHAGA fLWAVOACTER/UMOXIDASE - OXIDASE +

PERITRICHOUS POLAR PERITRICHOUSFLAGELLA FLAGELLA FLAGELLA POLAR FLAGELLA

ENTEROBACTERIACEAE CHOTOACTER/UM 40-50G 0-60% G-C

I ILUC/BACrER/UM V/BR/O AERONONAS

FIG. 1. Basic diagnostic scheme for marine gram-negative bacteria (adapted from reference 2).

investigators and in our laboratory (2, 3, 11). Strainsincluded in the major groups were subjected to nu-merical taxonomy analysis, with a total of 85 charac-teristics recorded for each strain. Pseudomonas, Cy-tophaga, Flavobacterium, and Chromobacterium spp.were also isolated (11, 12, 23) but at a very low fre-quency of occurrence. The very few fungal isolatesobtained among the isolates were not identified tospecies level.

Reference cultures. Bacterial strains included inthe analyses as reference were: Vibrio parahaemoly-ticus strains FC1011 and 163, isolated by R. Colweland R. Sakazaki, National Institute of Health, Tokyo,Japan, respectively; strain PCP, a freshwater isolateprovided by D. McKay and C. R. Jenkin, Universityof Adelaide, Adelaide, Australia; Vibrio fischeri, fromCarolina Biological Supply Co., Gladstone, Ore.; Aero-monas liquefaciens (A. hydrophila), provided by Pe-ter K. Chen, Biology Department, Georgetown Uni-versity, Washington, D.C.; and Vibrio cholerae, a non-choleragenic strain obtained from James Hawley, An-alytab Products Inc.Computer analyses. The methods for numerical

taxonomy were those routinely employed in our labo-ratory. The final matrix included 93 strains and 85characters. The data were analyzed using the simplematching coefficient (SsM) (22), which includes bothpositive and negative matches, and the Jaccard coef-ficient (Si) (23), which excludes negative matches.Programs used, including the UMTAXON 3 and1SP53 program packages, are available on the Univer-sity of Maryland UNIVAC 1108 computer.

Serology. Selected isolates, resembling V. para-haemolyticus as defmed (16), were serotyped withanti-K serum. Selected isolates resembling Vibrio an-guillarum (2, 5) were serotyped with V. anguillarumgroup antiserum.

RESULTSThe station locations and the total viable

counts of bacterial heterotrophs and vibrio-like

organisms isolated from water samples fromwhich copepods were collected are given in Ta-ble 1. Plate counts were 3 or less colony-formingunits per ml on both the 2216 and TCBS agar.The source of the copepods recorded by species,along with comparative data on total viablecounts of heterotrophic bacteria versus vibrio-like organisms isolated from the external surfaceof copepods and from gut-surface samples, aregiven in Table 2. The latter could not be deter-mined accurately as being of gut origin alone,because of the nonselective procedure for isola-tion of the bacteria (see Materials and Methods),and so the term gut-surface isolate was used.Comparison of data presented in Tables 1 and 2shows that many more bacteria were found as-sociated with copepods than were found to befree-living in the water column, indicating apreferential adsorption to or association of thebacteria with the animals. Fewer bacteria wereisolated from gut surface samples than from thecopepod surface samples (Table 1).The taxonomic distribution ofmicroorganisms

in water samples from laboratory dishes in whichnormal, healthy copepods were reared, in prep-arations of moribund animals reared in the lab-oratory and aboard ship, and in estuarine waterand ocean water samples from the stations atwhich the copepods were collected is given inTable 2. Of the 329 strains examined, Vibrio spp.were the most abundant of the bacterial taxapresent in the pelagic samples, with Pseudomo-nas spp. the most abundant in laboratory-reared, healthy copepods. The bacterial generarepresented in water samples from moribundcopepods reared in the laboratory or in ship-board culture dishes were similar.

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Data for estuarine, pelagic, and laboratory-reared animals are given in Table 3, showingbacterial strains isolated from water, from thesurface of copepods, and from the copepod gutsurface samples. The estuarine copepods were

found to have the highest representation of Vi-brio spp., with laboratory-reared and pelagic

animals sharing a lower count for the vibrios. Onthe other hand, the pelagic copepods possessedmore Pseudomonas spp. in the gut flora thancopepods collected from the estuarine environ-ment or copepods reared in the laboratory.The distribution of bacterial genera compris-

ing the associated or commensal flora of the

TABLE 1. Total viable counts ofmarine heterotrophs and Vibrio-like organisms recovered from surfaceversus gut-surface samples from copepods collected during cruise

Surface' Gut-surface'Copepod Source

Heterotrophs Vibrio Heterotrophs Vibrio

Acartia sp. Anclote R., Fla. 60 3 NDAcartia sp. Anclote R., Fla. ND 1 x 103 26Pleuromamma sp. Station 1 ND NDPleuromamma sp. Station 1 ND 3 x 102 0P. regalis Station 1 1 x 103 13 4.7 x 102 3L. aestiva Station 2 1 X 103 1.2 x 102 1.37 x 103 70L. aestiva Station 3 3 x 102 30 1.4 x 102 16L. aestiva Station 3 3 x 104 2.9 x 103 1 X 105 28C.furcatus Station 3 >100 25 1 x 105 1 X i05Acartia sp. Station 3 1 x 103 1 X 103 1 X 103 1 X 103L. aestiva Station 4 3 x 102 <1 20 <1Acartia sp. Station 4 102 <1 1.4 x 102 <1L. aestiva Station 4 20 <1 23 3

a Total counts of heterotrophs were made on marine agar 2216; total counts of Vibrio-like organisms weremade on TCBS agar. Counts are average of three animals. ND, Not done.

TABLE 2. Taxonomic distribution of microbial isolatesNo. (% of total) of microbial isolates from source:

Laboratory- Moribund lab- Moribund co-Microorganisms reared, nor- oratory-reared pepods in cul- Estuarine Pelagic waterreaed,nor ortor-rered ture aboard water eacwtrmal copepods copepods shr=b1p (N=w8) (N = 39)(N= 12)a (N-= 19) (N- 11) 8

Vibrio spp. 2 (16.7) 10 (52.6) 5 (45.5) 8 (100) 29 (74.3)Pseudomonas spp. 9 (75.0) 8 (42.1) 5 (45.5) 0 (0) 3 (7.7)Cytophaga/Flavobacterium Spp. 1 (8.3) 1 (5.3) 0 (0) 0 (0) 4 (10.3)Chromobacterium Spp. 0 (0) 0 (0) 1 (9.0) 0 (0) 0 (0)Fungal species 0 (0) 0 (0) 0 (0) 0 (0) 3 (7.7)

N, Number of strains included in the analysis.

TABLE 3. Taxonomic distribution of the microbial flora of estuarine, pelagic, and laboratory-rearedanimals"

Estuarine samples Pelagic samples Laboratory(N= 54) (N= 135) reaNed

MicroorganismsSur- Gut- Sur- Gut- Gut-

Water face sur- % Water face sur- % sur- %face face face face face

Vibrio spp. 8 22 21 94.4 29 41 15 63.0 29 82.9Pseudomonas spp. 0 1 0 1.9 3 12 25 29.6 4 11.4Cytophaga/ 0 1 0 1.9 4 0 2 4.4 2 5.7Flavobacterium spp.

Chromobacterium spp. 0 1 0 1.9 0 0 0 0.0 0 0Fungal species 0 0 0 4 0 0 3.0 0 0

a Water samples, copepod surface washes, and combined gut-surface samples are shown. A total of 224 strainswere included in the analysis. N, Number of strains in each analysis.

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754 SOCHARD ET AL.

copepod species sampled during this study isgiven in Table 4. One striking fmding was thatwhen samples of 23 laboratory-reared animalswere collected aseptically and cultured, all of theisolates were Vibrio spp. (see Table 3). Bacterialstrains obtained from all the animals studiedwere Vibrio and Pseudomonas spp., with Vibriospp. being three times more abundant.The overall distribution of bacterial genera

represented among strains comprising the bac-terial flora ofcopepods is given in Table 5. Vibriospp. clearly were the most abundant of the bac-terial species found associated with the copepod,with Pseudomonas spp. the next most abundant.Fungal species were rarely encountered and onlyon pelagic copepods.


Of the Pseudomonas spp., a large proportionwere Pseudomonas fluorescens. Of greatest in-terest was the large number of vibrios foundassociated with the copepod. The vibrios werefurther tested for numerical taxonomy analysis.The S-value matrix computed for the Vibrio

isolates, including the reference strains of thegenus Vibrio, is shown in Fig. 2. The sortedsimilarity matrix is based on the SJ coefficient.Eight groups emerged from the data analyses.Figure 3 provides a simplified dendrogram, pre-pared using the SJ coefficient, with the eightgroups of vibrios represented as phena. Similar-ity (S) for the eight phena was at the 75% level.Table 6 presents the characteristics, or posi-

tive responses, as percentage of the unit char-

TABLE 4. Distribution of bacterial genera associated with species of copepodsa

Copepod species [ Source ] Surface J Gut-surface Asection | Fecal pellets

A. tonsa

L. aestiva

P. regalis

Pleuromamma sp.

C. furcatus

Anclote R.(estuarine)

MIZARdStation 3(pelagic)

MIZARStation 4(pelagic)

BayboroHarbor(estuarine)

Laboratoryreared







15 Vibrio app.1 Chromobacterium sp.

5 Vibrio app.

2 Pseudomonas spp.

7 Vibrio spp.1 Cytophaga orFlavobacterium sp.

5 Pseudomonas spp.2 Vibrio spp.

11 Vibrio spp.2 Pseudomonas spp.5 unknown'

3 Pseudomonas spp.1 unknownc

No growth

No growth

8 Vibrio spp.1 Pseudomonas sp.

7 Vibrio app.1 Pseudomonas Sp.1 unknownc3 Vibrio spp.

3 Pseudomonas app.1 unknown'

15 Vibrio spp.1 unknown'

9 Vibrio spp.10 unknownsc9 Pseudomonas app.1 Cytophaga orFlavobacterium sp.

8 Vibrio spp.2 Pseudomonas spp.

13 Vibrio spp.7 Pseudomonas spp.2 Cytophaga orFlavobacterium spp.

2 unknownc3 Pseudomonas spp.

5 Pseudomonas Spp.4 Vibrio app.

6 Pseudomonas spp.

2 Vibrio app.2 Pseudomonas Spp.2 unknownc

23 Vibrio spp.e

3 Pseudomonas app.

a Samples are from a three-animal composite sample except for aseptic gut dissections (see the text).-, Not done.

c Isolate not viable on subculture.d MIZAR, Cruise of U.S.N.S. MIZAR.'A total of 23 animals were sampled.

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TABLE 5. Distribution of bacterial genera among329 strains isolated from copepods

Organism No. Percent

Vibrio spp. 182 55Pseudomonas spp. 73 22Cytophaga or Flavobacterium 11 3

spp.Chromobacterium spp. 2 <1Unidentifieda 55 17Fungi 6 2

a Strains not viable on subculture or not identifiedto genus and/or species.

acters for phena 1 to 8.All of the bioluminescent vibrios grouped in

phenon 1, which, incidentally, excluded the sin-gle luminescent reference strain, V. fischeri. Thebioluminescent isolates were examined further,employing additional biochemical tests as wellas electron microscopy. Phenon 1, comprisingthe bioluminescent strains, grew well on TCBSagar and, by electron microscopy, possessed asingle polar flagellum. These strains grew atboth 37 and 50C. Unfortunately, this group couldnot be identified to any of the species describedby Hendrie et al. (11).Very few of the isolates could be serotyped.

Of these, two isolates, 33 and 108, were typablewith anti-V. parahaemolyticus K sera K-18 andK-19. Strain 108 clustered with phenon 3, whichcontained the reference strains of V. parahae-molyticus. Strains 124 and 125 reacted with anti-V. anguillarum serum. Strain 124 fell intophenon 6, whereas strain 125 was lost beforesufficient data could be gathered for computerassignment.

Figure 4 shows typical adult male and adultfemale A. tonsa Dana reared in captivity. Fe-males are generally larger and more plump thanmales, being adapted to carrying eggs. The cau-dal rami may easily be observed.

DISCUSSIONIt has been concluded by many investigators

that, like many, if not all, terrestrial animals, thedigestive tracts of most marine animals containa commensal microbial flora (1, 6, 8, 9, 20, 24).Exceptions have been recorded, with results oflight and scanning electron microscopy provid-ing evidence for the absence of an associatedmicrobial flora in the gut of the crustacean spe-cies Limnoria, Chelura, and Oniscus spp. (3, 18,19). However, in the study of the copepod, azooplankton member, the presence of a com-mensal flora, comprising several genera of het-erotrophic bacteria in the digestive tract of sev-eral species ofmarine copepods, i.e., crustaceans,

is clearly established. Interestingly, after elimi-nation of fecal pellets by the copepods, it wasfound, on subsequent culture of the dissectedgut, that much lower numbers of bacteria werefound in the gut. Thus, the release of copepodgut flora bacteria via fecal pellets provides amechanism for the wide distribution of thesebacteria in the water column and sediment ofestuaries and the oceans.The significant number of marine vibrios

found to be associated with marine copepods isindeed noteworthy, as is the monotypic flora inthe gut of the copepod A. tonsa, maintainedunder defined laboratory conditions in the lab-oratory. Although culture conditions for the co-pepod have been discussed elsewhere (25, 26), itis well to reiterate some of the details. Copepodsin culture through multiple generations aremaintained in Millipore-filtered water, i.e., sea-water is passed through a bacteria-retaining Mil-lipore filter before entering the aquaria, andfood, in the form of three species of bacteria-freealgal preparations, is also provided aseptically,since the algae are cultured aseptically underartificial lighting with temperature and otherphysical parameters carefully controlled (19,20).Thus, a unique association between bacteria andcopepods appears to establish itself. The uniquegut isolates have not separated, or grouped inthe computer analyses. Thus, they are referredto as "marine vibrios."

It is conceivable that maintaining A. tonsa inlaboratory culture imposes special restrictionson the expression of the normal flora, with theresult that selective pressures are imposed bythe artificial conditions of the laboratory envi-ronment. For example, A. tonsa are in natureherbivores. The choice of three algal forms for

% SIMILARITY NO. OF IDENTITYSTRAINS

15 "Morine-vibros"

7 "Marne-vibrios""Morine - vibrioMarine -vabrio"

2 V,br,o parohoemo/yhcasVibr,o sp,V,brio cho/oroe

15 "Morine vibris'"Marine - vibro'

5 Morine - vibros"

More - vibrios"

7 "Marine - vibroos"Morine vibrio"

23 Morine - vibrios"Acart,o tongo gut

Aeromonos hyd&ophio"Marine- vibrio"

PHENON

2

3

4

5

6

7

FIG. 3. Simplified dendrogramprepared using theSi coefficient and average linked method of cluster-ing.

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TABLE 6. Summary of the characteristics of the phena obtained in the analyses, expressed as percentpositive responses

PhenonCharacteristic

1 2 3 4 5 6 7 8

SourceSeawaterCopepod

Colonial morphologySmall colony, 1-mm diameterSpreaderOpaqueTranslucentOff-whiteBioluminescent

MicromorphologyCurved rodsCoccobacilliMotile

BiochemistryArginine dihydrolaseLysine decarboxylaseOrnithine decarboxylaseFermentative metabolismIndole productionN03-NO2O-Nitrophenyl-/?-D-galactopyranosideOxidaseTCBS, alkalineTryptophan deaminaseVoges-Proskauer reaction

Degradation of:ChitinGelatinLecithinTween 20Tween 40Tween 60Tween 80Urea

Sensitivity to:0/129AureomycinBacitracinChloramphenicolDihydrostreptomycinErythromycinNovobiocinPenicillinPolymyxin BTerramycinTetracycline

Growth at 43°CGrowth in:3% (wt/vol) NaCl7% (wt/vol) NaCl10% (wt/vol) NaCl

Utilization of:AmygdalinArabinoseMannoseSaccharoseSodium citrate

27 073 86

13 860 0

100 147 860 10067 0

7 053 71100 100

7 0100 100100 100100 100100 100100 10060 2987 10073 860 00 0

100 100100 100100 100100 100100 100100 100100 10020 14

0 7 20 180 93 80 82

0 0 0 00 87 0 0

100 0 0 00 100 100 100

100 100 100 1000 0 0 0

50 0 0 050 93 0 91100 100 100 100

0 0 0 36100 100 0 0100 0 0 0100 100 100 100100 100 100 100100 100 100 1000 7 100 0

100 100 100 82100 20 60 1000 87 100 910 100 20 73

0 93 100 50100 100 80 100100 100 100 100100 100 100 100100 100 100 100100 100 100 100100 100 100 10050 0 0 0

7 14 50 0 60 9140 0 100 0 100 640 0 100 57 0 0

100 100 100 100 100 100100 100 0 100 100 10080 100 100 71 100 82100 100 0 100 100 910 0 100 0 75 920 50 100 14 100 9160 100 100 86 100 10060 100 100 93 100 1007 0 100 67 0 0

100 100 100 100 100 100100 100 50 100 20 910 0 0 67 0 0

100 100 100 100 80 640 14 0 7 0 093 86 100 87 100 8213 29 0 73 60 0

100 100 100 100 0 82

0 0100 100

0 1000 00 0

100 100100 1000 0

29 10057 100100 100

0 640 00 0

100 100100 100100 100100 10071 10086 1000 029 0

14 100100 100100 100100 10086 100100 100100 1000 0

29 100100 800 0

100 10071 2086 100100 10086 10071 100100 10086 1000 0

100 100430 0

0 00 00 00 10014 0

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4

I mmFIG. 4. Typical male and female specimens of A. tonsa Dana. The females, adapted for egg bearing, are

typically larger than the males. The latter are further differentiated by their longer, slightly more curvedantennae.

laboratory "fodder" was a conscious choice foroptimum propagation. In the wild, the animalscome into contact with many bacterially con-taminated algal forms also serving as food (8).

Interestingly, the flora of A. tonsa organismsisolated during cruises, from estuarine samples,and from normal as well as moribund copepodsrevealed that the bacterial flora does not varysignificantly.The gut flora organisms formed a cohesive

group at S = 60 to 100%. The A. tonsa gutisolates can be seen in Fig. 2 and 3 as phenon 8.Reference strains included in the computer anal-ysis grouped separately with no observable re-lationship with the fresh isolates. V. fischeri, forexample (Fig. 2), grouped separately from bio-luminescent isolates. However, phenon 2 may benonluminescent strains of V. fischeri, if onechose to group phena 1 and 2 as V. fischeri.Two strains of V. parahaemolyticus, strains

FC 1011 and 163, grouped as phenon 3, againseparate from the marine isolates under study.V. cholerae (non-choleragenic strain) and Vibriostrain PCP (a freshwater isolate) were separatedfrom the marine vibrios at the species level. A.liquefaciens (A. hydrophila) was not related toany of the marine Vibrio spp. of the study.

Several strains of Photobacterium phospho-reum were examined during the research workreported here. These bacteria were relativelyeasy to distinguish from the marine vibrios, sinceP. phosphoreum could be isolated from the lightorgans of deep-sea fish and required lower in-cubation temperatures. The characteristic lightemitted by them was of a different wavelengthfrom that of the marine bioluminescent vibriosfound in this study, i.e., phenon 1 (see Fig. 2 and3).

Several aspects require further study. It is notknown what ecological parameters affect the gutflora of the copepods when they are brought intolaboratory culture. The commensal bacterial

flora does not vary much from the wild. How-ever, differences, possibly associated withchanges in salinity, were observed in the flora ofpelagic versus estuarine versus laboratory-reared copepods (Tables 2 and 3).Such differences in the bacterial flora in oth-

erwise similar copepods collected from differentlocations can be speculated to arise from feedinghabits, since only A. tonsa is truly herbivorous,the others being omnivorous.

Significant association of bacterial specieswith copepod species was not observed.A very difficult problem is the taxonomy of

marine vibrios. Only a few Vibrio spp. are avail-able in culture collections, yet vibrios often arethe dominant organisms in the marine and es-tuarine environment. V. cholerae, V. parahae-molyticus, V. alginolyticus, V. anguillarum, V.fischeri, and V. metchnikovii are the only rela-tively completely characterized species (14).From the taxonomic results obtained in thisstudy, it is clear that there are several newspecies of marine Vibrios that will need to bedescribed and named.

ACKNOWLEDGMENTM.R.S. was an NRC-NRL Resident Research Associate at

the Naval Research Laboratory, Office of Naval Research,Washington, D.C. 20375.

LITERATURE CIWED1. Alexander, M. 1971. Microbial ecology. John Wiley and

Sons, Inc., New York.2. Bain, N., and J. M. Shewan. 1968. Identification of

Aeromonas, Vibrio and related organisms, p. 79-84. InB. M. Gibbs and D. A. Shapton (ed.), Identificationmethods for microbiologists, part B. Academic Press,New York.

3. Boyle, P., and R. Mitchell. 1978. Absence of microorga-nisms in crustacean digestive tracts. Science 200:1157-9.

4. Brenner, S., and R. W. Horne. 1979. A negative stainingmethod for high resolution microscopy of viruses.Biochim. Biophys. Acta 34:103-110.

5. Carpenter, K. P., J. M. Hart, J. Hatfield, and G.Wicks. 1968. Identification of human vibrios and allied

758 SOCHARD ET AL.

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organisms, p. 9-18. In B. M. Gibbs and D. A. Shapton(ed.), Identification methods for microbiologists, partB. Academic Press, New York.

6. Colwell, R. R., and J. Liston. 1962. The natural bacterialflora of certain marine invertebrates. J. Insect Pathol.4:23-33.

7. Colwell, R. R., and W. J. Weibe. 1970. "Core" charac-teristics for use in classifying aerobic heterotrophicbacteria by numerical taxonomy. Bull. Ga. Acad. Sci.28:165-185.

8. Gophen, M., B. Z. Cavari, and T. Benman. 1974. Zoo-plankton feeding on differentially labeled algae andbacteria. Nature (London) 247:393-394.

9. Harding, G. C. H. 1973. Decomposition of marine cope-pods. Limnol. Oceanogr. 18:670-873.

10. Heinle, D. R. 1969. Culture of calanoid copepods in syn-thetic sea water. J. Fish. Res. Bd. Canada 26:150-153.

11. Hendrie, M. S., W. Hodgkiss, and J. M. Shewan. 1970.The identification, taxonomy and classification of lu-minous bacteria. J. Gen. Microbiol. 64:151-169.

12. Hendrie, M. S., T. G. Mitchell, and J. M. Shewan.1968. The identification of yellow pigmented rods, p.67-78. In B. M. Gibbs and D. A. Shapton (ed.), Identi-fication methods for microbiologists, part B. AcademicPress, New York.

13. Hendrie, M. S., and J. M. Shewan. 1966. The identifi-cation of certain Pseudomonas species, p. 1-7. In B. M.Gibbs and F. A. Skinner (ed.), Identification methodsfor microbiologists, part A. Academic Press, New York.

14. Lee, J. V., T. J. Donovan, and A. L. Furniss. 1978.Characterization, taxonomy, and emended description

of vibrio metschnikovii. Int. J. Syst. Bacteriol. 28:99-111.

15. Leifson, E. 1951. Staining, shape, and arrangement ofbacterial flagella. J. Bacteriol. 62:377-387.

16. Miwatani, T., and Y. Takeda. 1976. Vibrio parahae-molyticus. A causative bacterium of food poisoning.Saikon Publ. Co. Ltd., Tokyo.

17. Murchellano, R. A., and C. Brown. 1970. Heterotrophicbacteria in Long Island Sound. Mar. Biol. 7:1-6.

18. Ray, D. L. 1959. Proc. Am. Wood Preserv. Assoc. 55:147.19. Ray, D. L., and J. R. Julian. 1952. Occurrence of cellu-

lase in Limnoria. Nature (London) 169:32-33.20. Savage, D. C. 1977. Microbial ecology of the gastrointes-

tinal tract. Annu. Rev. Microbiol. 31:107-133.21. Skerman, V. B. D. 1967. A guide to the identification of

the genera of bacteria. The Williams and Wilkins Co.,Baltimore.

22. Sneath, P. H. A. 1956. Cultural and biochemical charac-teristics of the genus Chromobacterium. J. Gen. Micro-biol. 15:70-98.

23. Sneath, P. H. A., and V. G. Collins. 1974. A study intest reproducibility between laboratories. Antony vanLeeuwenhoek, J. Microbiol. Serol. 40:481-527.

24. Steele, J. H. 1974. The structure of marine ecosystems.Harvard University Press, Cambridge, Mass.

25. Wilson, D. F., and K. K. Parrish. 1971. Remating in aplanktonic marine calanoid copepod. Mar. Biol. 9:202-204.

26. Zillioux, E. J., and D. F. Wilson. 1966. Culture of aplanktonic calanoid copepod through multiple genera-tions. Science 151:996-998.

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