Vol. 54,No. 1 APPLIEDANDENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 1-9
0099-2240/88/010001-09$02.00/0
Copyright C 1988, American Society for Microbiology
In
Situ Survival
of Vibrio cholerae and Escherichia coli in
Tropical
Coral
Reefs
NERYBELLEPEREZ-ROSAS ANDTERRY C. HAZENt*
MicrobialEcology Laboratory, Departmentof Biology, College of NaturalSciences, University ofPuertoRico, RioPiedras, Puerto Rico00931
Received 14 May 1987/Accepted6October1987
Vibrio choleraeand Escherichia coliwereinoculated intomembranediffusionchambers and placed around twosmallcoralreef islandsin Puerto Rico and monitored for5days. Several chamberswerealsoburied in the
sandsofoneofthereefs.Both E. coli and V. cholerae densities declined by 2 orders of magnitude,asmeasured
by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the
density of neitherbacteria changed dramatically when thesamesampleswereanalyzed by epifluorescent direct
counts. Differencesin the two directcountmethods were accounted for by changes in cell morphology that
occurredin bothbacteriaafterexposureto seawater. Morphological changes occurredmorerapidly inE.coli
comparedwiththosein V.cholerae.Bacteria in chambers exposedtosediment didnotshow significant changes in morphology and had onlyaslight decline in density. Physiological activity declined bymorethan 40% for
both bacteriawithin 24 h.Thedecline in activitywaslessseverein the sediments. Tropical coralreef sands and
turtlegrassbedswereshowntobe lessstressful environments for V. cholerae and E. coli than would have been predictedfromtemperateand microcosm studies. V. choleraecansurvive the in situ conditionsofa tropical
coralreef and could becomea sourceofbacterial contamination for fish and shellfish in thisenvironment.The simultaneous monitoring ofE. coli levels established that this bacteriacan not beusedas an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments putonE. coli. Both bacteria could beofgreater public health importance in tropical marine
areasthanpreviously imagined.
Itis known that Vibrio cholerae and other vibrios are part
of the indigenous microflora of most estuaries (8, 18).
Consideringthe reportsof recent outbreaks of cholera traced to shellfish from warm waterenvironments, it has become
increasingly important to understand the ecology of these
bacteria. Many studies have suggested that V. cholerae is
incapable ofsurviving in seawater at salinities of 35%o and that high temperatures, i.e.,
>250C,
are stressful (17, 22). However, recent studies have suggested that V. cholerae and Escherichia coli, its indicator, may be able to survive under theseconditionsorunderstarvationconditions and be unculturable on standard media (3, 8, 26). It has also been well documented that the survival of E. coli can bedramat-ically decreasedby high solarradiation(11, 20). Since all of theseconditions arenormal and constant intropical marine areas like Puerto Rico, it was surprising to us that our
laboratory(4, 10, 15, 16, 24) and others (12, 13, 22) were able to isolate V. cholerae and E. coli from tropical coastal waters.Other work by our group had shown that Aeromonas
hydrophila was capable of growing at densities above
107
cellsper mlwhendiffusionchambers with thebacteriawere
suspended in marine coastal areas ofPuerto Rico contam-inatedwithrumdistillery effluent(4). We also observed that rum distillery effluent served as a chemoattractant for V. cholerae (10). Thus, we undertook the present study to determine V. cholerae andE. coli survival by usingin situ diffusionchambersatseveralmarine sites andbyemploying
* Correspondingauthor.
tPresent address: Environmental Sciences Division, Savannah River Laboratory, E. I. Du Pont de Nemours & Company, Inc., Aiken, SC29808.
twodirect count methods and two populationactivity mea-surements. Previous studies had used laboratory micro-cosms and had notmeasuredphysiological activity (18). The presentstudy was undertaken toestablish whether V. chol-erae could survivethe in situ conditionsofatropical coral reef and thus whether this environment could become a reservoir source for fish and shellfish in coral reefs. The simultaneous monitoring ofE. coliestablished whether this
bacteriumcouldbe used as anindicator ofV.cholerae in the coral reef environment.
(This study was partofthe M.S. thesis of N.Perez-Rosas
at theUniversity ofPuertoRico, RioPiedras, PuertoRico, 1983.)
MATERIALS ANDMETHODS
Study sites. Palominitos is a coral sand island off the northeastern shoreofPuertoRico,
180
20'N,65025' W(Fig.1). It hasawell-developedcoral reef dominatedbyAcropora
palmata and Montastrea annularisalongtheeast
side,
with turtle grass beds(Thalassia testudinum)dominating
thewestside. This island is
frequented
for recreationon weekends. LaGata Islandislocatedontheinnershelf ofthesouthwestcoastofPuertoRicoat17°57' Nand67002' W near thesmall
fishing village of Parguera (Fig. 1).Theislandwascreated
by
apatch reef, with subsequent colonization
by
theredman-grove Rhizophora mangle. The south part ofthe island is dominatedbyacoral reef dominatedbyM.annularis andA.
palmata (1). The north side is dominated
by
extensive beds ofturtle grass. During 1980, La Gatawasinaugurated
as arecreational areaby the Departmentof Natural Resources.
During 1981, atotal of85,909persons visitedthe islandfor
1
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FIG. 1. Map ofstudy sites atLaGata and PalominitosIslands,PuertoRico.
anaverage of7,160 persons monthly and 239 persons daily
(Department of Natural Resources, personal
communica-tion). The installation oftwo toilets created a raw sewage outfall on the island. Dye studiesby our laboratoryshowed
that 20 min after a dye cone was flushed dye could be detected in the surrounding waters. After 1 h, the dye had
spread to 50% of the water along the north coast ofthe
island, and within 2 h it coveredthe wateralong the entire north coast, includingthearea reservedforswimming.
Water analysis. Measurements were taken in situ for
conductivity, salinity,pH, dissolved oxygen, light intensity, and temperature. The pH was measured with a digital pH meter(model 201; Orion Research, Inc., Cambridge, Mass.), anddissolved oxygen was measured with a DO meter (model 57; Yellow Springs Instruments Co., Yellow Springs, Ohio). An S-C-T meter (model 33; Yellow Springs) was used to measure conductivity and salinity. Turbidity, alkalinity, hardness, and ammonia measurements were done in the field by using a spectrophotometer (Mini Spectronic 20; Bausch & Lomb, Inc., Rochester, N.Y.). Light intensity was mea-sured in the field with an underwater photometer
(Proto-matic, Dexter, Mich.). For chlorophyll a determination, watersamples were placed in amber-colored plastic bottles andanalyzedatthe laboratory by thetrichromatic extraction method (2). Other samples were fixed with mercuric
chlo-ride,sulfuric acid, and zinc acetate before being transported
tothe laboratory, wheretheywereanalyzed fornitrateplus nitrite, sulfate, total phosphorus, and orthophosphate,
ac-cording to the procedures in StandardMethods
for
Water andWastewaterAnalysis (2).Bacteriologicalanalysis. Directcellcountsfor V.cholerae and E. coli in diffusion chambers were done
by using
aCoulter Counter (Coulter Electronics, Inc.,
Hialeah,
Fla.)
and acridine orange staining(AODC). Red-fluorescingcells are assumed tobe active in protein synthesis since the red fluorescence is caused by a dominance in RNA content. Cells with more DNA than RNA will fluoresce green (9). Studies in our laboratoryhave shown thatby careful prep-aration of reagents E. coli and other bacteriacan be mea-sured for their relative activity in this way (7, 19). Total number ofbacteriaand the number involved in respiration
weredetermined by thetechnique of Zimmermanetal.(28).
Survival studies. Forsurvival studies, Plexiglas (Rohm& Haas Co.,
Philadelphia,
Pa.) diffusion chambers, amodifi-cation of those of McFeters and Stuart (21), with 100-ml
capacities were used with 0.45-,um-pore-size,
nylon-rein-forcedVersapor membrane filters (GelmanInstrument
Co.,
AnnArbor, Mich.)asdiffusionsurfaces (4,14).
0-rings
were addedtothechamberstoreduceleakageandcontamination.Pure cultures of V. cholerae andE. coliwere grown in 5% tryptic soy broth at 37°C for 24 h. The cells were then harvested by centrifugation (10,000 x g for 10 min) and
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V. CHOLERAE AND E. COLI IN CORAL REEFS 3
TABLE 1. Water quality of LaGata Island and Palominitos Island, Puerto Rico'
Temp(°C) Dissolved Salinity Nitritesplus Ortho- Total Chlorophylla
Siteb oxygen pH MO nitrates phosphate phosphorus
(mg/liter)
Air Water (mg/liter) (mg/liter) (Lg/liter) (pg/liter)
LG-N 24.3 + 0.6 26.2 ± 0.2 7.1 ± 0.3 7.2 ± 0.2 34.2± 0.5 0.2 ± 0.0 0.7 ± 0.5 0.9± 0.7 0.63 ± 0.60
LG-S 24.9 ±0.4 26.1 ± 0.2 7.5 ± 0.2 7.7 ± 0.1 33.6 ± 0.5 0.3 ± 0.0 0.7 ± 0.1 0.8 ± 0.8 0.80± 0.04 PI-T 21.4 ± 0.5 20.5± 0.5 8.2 ± 0.1 7.1 ± 0.1 34.0 ± 0.0 0.1 ± 0.0 <0.1 0.1 ± 0.0 0.19± 0.03 PI-C 21.4 + 0.5 19.8 ± 0.6 7.9± 0.1 7.4 ± 0.2 34.0 ± 0.0 0.1± 0.0 <0.1 0.1 ± 0.0 0.27± 0.08
'All values aremean+ 1 standard error (n, >5).
bLG-N,LaGata north;LG-S,LaGatasouth; PI-T, PalominitosIsland,Thallassia site; PI-C, Palominitos Island, coral.
suspended in filter-sterilized phosphate-buffered saline (pH
7). Cell density was determined with a model ZF Coulter Counter andadjustedto aconcentration of107cells perml. The bacterialsuspension was placed into the sterile diffusion chamberjust before it was placed at the study site. At the
study site,atotal offivechambers were placed strategically at a depthof1 m. Periodically, 1.0-ml samples were taken
from each chamber witha sterile syringe. Of each sample, 0.5mlwasfixed with1.5mlof phosphate-buffered Formalin
Z
E
0
0 12 24 36 48 60
Time(hr)
for latercountingatthelaboratory withaCoulter Counteras described by Hazen and Esch (14). The other 0.5 ml was incubated with INT and fixed by the method of Zimmerman etal. (28). Thepreserved samplewasthen storedonice for membrane filtration at the laboratory and subsequent total directcountsandactivitymeasurements asdescribed above. Five chambers were also carefully buried in the sandy sedimentnearthe chamberssuspended in thewatercolumn.
To avoid unnecessary disturbance, two chambers were
72 84 96 108 2; 10' 106 = ~ ~~~~*VC site s-09EC SiteS _ O~~~~~EC Site S-0 12 24 36 48 60 72 84 96 108 Time(hr)
FIG. 2. Changesintotaldensityfor V. cholerae(VC)and E. coli(EC),asmeasuredbyCoulter Counter(A)and AODC(B),at La Gata Islandby site (mean + 1 standarderror, n = 4).Sitesaredefined in Table1,footnoteb.
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60 40 20 0 12 24 36 48 60 72 84 96 108 Time(hr) 60 I I * QVCSiteN B 50 VC
sitesS
E EC Site N 40COEC
Site S aR 20 10 0 0 12 24 36 48 60 72 84 96 108 Time(hr)FIG. 3. Changes in percent activity forV. cholerae (VC) andE. coli(EC), as measuredby AODC(A), and inpercent respiration,as measuredbyINT(B),atLaGataIsland bysite (mean t 1 standarderror,n =4). Sitesaredefined in Table 1,footnoteb.
removed and sampled at 24h, andthe remaining chambers were removed and sampled at 42 h. All sampling and measurements were as describedabove.
Data analysis. The data wereanalyzed byusing prepared programsfor AppleII,Macintosh,and IBM 4321 computers.
Factorial analyses ofvariance were used to test for differ-encesamongsitesandcollectiontimes. Dataweresubjected
to theappropriate transformation before statistical analysis bythe method ofZar(27). Anyprobabilityless thanorequal to 0.05wasconsidered significant.
RESULTS
Water quality. The two sites at La Gata Island were not
significantlydifferent in water quality (Table 1). All param-eters measured were stable during the 108-h sampling pe-riod. The same observations were made at the two sites at
Palominitos Island(Table 1).
Survival ofbacteria insitu.At LaGata Island, the densities
ofV.choleraeasdetermined by Coulter Counter counts (CC
counts) in the diffusion chambers decreased
significantly
overtime(F= 47.0,df= 17and30,P = 0.001),
decreasing
by80%duringthe first 12h,andweresignificantly
higheratthe north site (F= 9.91,df= 1 and30, P = 0.001)(Fig. 2),
i.e., more than 50% higher after 12 h. Densities of V.
cholerae, as determined by AODC, were also
significantly
differentovertime(F=22.0,df= 17and30,P<0.0001)but not by site (Fig. 2). However, densities of V. cholerae, as
measuredby AODC, decreasedbyonly 10% after12h.The
proportion of the V. choleraepopulationthatwasactive, as measuredby AODC,declined significantlyduringthe first 9 h(F =51.0, df= 17and 30,P<0.0001)andthen remained low; the north site had a significantly higher
proportion
of active cells (F=5.63, df= 17 and30,P<0.05)(Fig. 3).Theproportion of the V. choleraepopulationthatwasrespiring,
asdetermined byINT, also declinedsignificantly duringthe first 9 h (F = 2.17, df = 17and 30, P < 0.05) butwas not
significantlydifferentbysite(Fig. 3).Theinitialdecline
(first
3 h)in activity andrespiration wasgreater than
70%,
after this bothmeasurementsfluctuated between 1to30%without apparent pattern.Densities ofE. coli in thediffusion chambers at LaGata
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V. CHOLERAE AND E. COLI IN CORAL REEFS 5
Island, as determined by CC counts, declined significantly
overtime(F = 15.0, df = 17 and 34, P < 0.0001) (Fig. 2), withless than 1% remaining after only 12 h. The same was
also true when densities of E. coli were determined by
AODC,i.e., over timedifferencesweresignificant (F = 157,
df= 10 and 24, P < 0.0001) (Fig. 2). The activity of the E.
colipopulation declined by 70% in the first 3 h and then fluctuated between 5 to 40%, with a largevariability among
samples (Fig. 3). The percentage of the E. coli population
that wasrespiringchangedsignificantlyover time (F= 126,
df= 10and 24, P < 0.005) (Fig. 3). Respiration rate of the E.
coli cells declined significantlyonlyduringthe first3 h, i.e., more than30%.Duringsubsequentsamplings, the variability
inthese measurements was even greater for E. colithan it
was for V. cholerae. In comparison, V. cholerae had a
similar survival rate as did E. coli at La Gata Island.
However, unlike E. coli, V. cholerae had a significantly highersurvivalrate andactivity at the north site.
At Palominitos Island, the densities of V. cholerae, as
determined by CC counts, in the diffusion chambers
de-creasedsignificantlyover time (F=5.16,df=6and 18, P=
0.001); therewere nodifferences by site (Fig. 4). After 12 h,
densities of V. cholerae in the water had declined 80o. Chambers buriedin thesediments atthese sites showedno
significant changes overtime(Fig. 5). Densities ofV. chol-erae in the water, as determined by AODC, showed a
significant increase, >20% after42 h (F= 3.50,df= 6and
18, P < 0.05); however, chambers buried in the sediments showed a slight, though insignificant, reduction (Fig. 4 and 5). The proportion ofthe V. cholerae population that was
active,asmeasuredbyAODC, declined significantlyduring the first 18 h (F= 3.62, df = 6and 18, P < 0.05) but then
increased briefly to80% (Fig. 6). Activity in the chambers that wereburiedin the sedimentsdecreased slowly by only
30% duringtheentire period (Fig.7). Theproportion ofthe V. choleraepopulationthat wasrespiring,asdetermined by
INT, did not decline significantly at the Thalassia site but
declinedby more than40%at theother site(Fig. 6).
Densities ofE. coliin the diffusion chambers at Palomi-nitos Island, as determined by CC counts, remained
un-changed both in the water and the sediments (Fig. 4).
However, when densities of E. coli were determined by AODC a 20% increase was observed during the first 24 h, followed by a 90% decrease. Inthe sediments, the AODC densities ofE. coli decreasedbyonly 50%after 42 h(Fig.
5).
Theactivity ofthe E. colipopulation declined by 80%in the
I
I I ' I -1 E .5 108 a ._t £ 0 108 0 O vcSiteC * VC Site T H ECSitsC O ECSiTeT I I I I I I I II
0 6 12 18 24 Tme (hr) 0 6 12 18 24 Time(hr) A I I 30 36 42 48 30 36 42 48FIG. 4. Changesintotaldensity forV.cholerae(VC)and E.coli(EC),asmeasuredbyCoulterCounter(A) and AODC (B), for chambers inwater atPalominitos Islandbysite(mean + 1 standarderror,n = 4).Sitesaredefined in Table 1, footnote b.
xml=rwdtm VOL.54, 1988 I I I I I I m I .J6
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1096
_0
lg_
-OVC Site c-nd * VCSite T-ssd E ECSitsC-sod - o EC Site T-ud 107 l 0 6 12 18 24 Time(hr) 109 1 - OVCSit C-ud * VCSiteT-ssd -1 ECSite C-sd O ECSite T-sd 1071 0 6 12 18 24 Time(hr) FIG. 5.Changes
in totaldensity
forV.(EC), as measured by Coulter Counter
chambers insediments(sed)at Palominit 1 standarderror, n = 3). Sitesare define
first 18 h and remained less than 5% coliactivityin thechambers in these
but30%ofthe cellswere still active percentage of theE. coli population changedsignificantly over time (F=
<0.05)(Fig. 6). Respirationrateofti
significantly only duringthe first 12t
In comparison, V. cholerae had a s vival rate than did E. coliat Palomir unlikeE. coli, V. cholerae had signil and respiration rates at the Thalass survivedand maintainedhigherlevel ration inthe sedimentsthan in theon
DISCUSSION Both directcount methods showec V. cholerae densities at La Gata Simultaneously, densities ofE. coli showed a veryprecipitous decline tc initial density in only 12 h. At Pal neither bacteria declined more tha exposure for either direct count met
densityagaindeclinedmorerapidly th
bersburied inthe sedimentatPalomin
in density for either direct count me
42-h period. Xu et al. (26), using m
seawater at a salinity of 5%o and 25°C
E.coli AODCdensitiesdecreasedby. increased by 33%. They also obsi activity and culturable densities dec
' l § lI = increased for V. cholerae. When they used even
higher
A salinities (25%o), the E. coli AODC densities and activity
levels increased
slightly,
while V. cholerae densities and activity levels decreased slightly. For both bacteria theyobserved decreases in culturabilityatthishigher salinity. In our study using in situ exposure to higher salinities and higher water temperatures, changes in E. coli population density and activity were always greaterthan the changes that occurred in V. cholerae. Although our Palominitos results paralleled those ofXu et al. (26), the La Gata sites had a much greater effectonsurvival andactivity of both E. coli and V.cholerae. Theonlyother similarstudies
reported
from in situ chambers in subtropical waters are those of 30 36 42 46 Colwell et al. (8) which were done at Bimini Lagoon with E. coli. They reported that little change in AODC densities ' } _ occurred over an 18-day period, but population activity and B culturability declined dramatically after only 4 days. Thus, as the mathematical model ofSeidler and Evans (22) pre-dicted, higher salinities and temperatures should decrease the probability of finding V. cholerae in tropical environ-ments. Hood andNess(18) alsoreportedthat V. cholerae in Floridasurvived better than E. coli in microcosms contain-ing sterile sediment or water at 20°C and 20%o salinity. Contrary to the results of our study, they found that V. cholerae could not survive in laboratory microcosms in
- nonsterilesediment. Since ourchambers did not allow direct
sediment contact with the
bacteria,
thismight
account for 30 36 42 48 the differences observed. Thus, sediment interaction and otherfactorscanapparently haveasignificant impact on V. cholerae(VC) and E. coli cholerae survival as observed at Palominitos.(A) and AODC (B), for The differences between the AODC and CC counts for V. os Islandby site (mean ± cholerae and E. coliweregreater than 1 order ofmagnitude dinTable 1,footnote b. for La Gata Island. This observation isundoubtedly because of the morphological changes that occur in E. coli in the absenceof culturablechanges. Asreported by others (3, 18, thereafter (Fig. 6). E. 26),weobservedshortening and condensation of the bacte--diments also declined, rial cellsatboth La Gata sites, although change occurred at after 42 h(Fig.7). The aslowerrateforPalominitos andatan even slowerratefor that was respiring also chambersburiedinsedimentatPalominitos. After24hofin 3.53, df= 6and 18,P situ exposureatLaGata, 50%of the cells hadtransformedto ieE. coli cells declined micrococci, i.e., <1 ,um. Since the CC counts were done h, i.e., morethan
40%.
witha10-,um aperture, themicrococcicould not be detected;ignificantly better sur- with the Coulter Counter unlike results obtained with the nitos Island. However, AODC method.
ficantly higher survival Physiological activity of the bacteria in the diffusion via site. Both bacteria chambers at La Gata Island, as measured by AODC and Isof activityand respi- INT,declined by more than40% after only 3 h of exposure verlying waters. forbothbacteria.The present study showed that V.cholerae
activity was affected less rapidly than that of E. coli and could maintainalevelof activity that was higher than that of E. coli under some conditions, e.g., in sediments and tropi-d thelargest decline in cal sea grass areas. Since E. coli is generally classified as a
during the first 24 h. nonsurvivor in marine environments (5, 6, 23), it is not in adjacent chambers surprising that it is rapidly and severely stressed in tropical less than0.1% of the marine environments. Other studies have also shown that lominitos, densities of sediment interaction will greatly decrease thestressing effect Ln 50% after 42 h of that the marineenvironment has on E. coli (18). The differ-thod; however, E. coli ences observed for different tropical marine environments ianV.cholerae. Cham- aresurprising since most studies would indicate that both E. iitos showed no change coli and V. cholerae should be rapidly inactivated and thus thod during the entire notbe found in these environments. Indeed, it appears that
iicrocosms of artificial coral sands and sea grass beds could harbor V.choleraeand for 96h, showed that even E. coli for much longer periods than previously
50%,whileV.cholerae thought. This suggests that the fecal coliform and fecal erved that population streptococci maximum contaminant levels used for marine reased for E. coli and recreational waters (6) are inadequate for tropical areas.
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V. CHOLERAE AND E. COLI IN CORAL REEFS 7 ~*60 A 40 20 0 0 6 12 18 24 30 36 42 48 Time(hr) 50 'B-O VCSiteC * VCSiteT 40 ^ ECSite C
om
ECSitT c 30 20 10 0 0 6 12 18 24 30 36 42 48 Time(hr)FIG. 6. Changes in percentactivity for V. cholerae(VC) and E. coli(EC), as measuredbyAODC (A), and in percent respiration, as
measuredby INT (B), forchambers inwater at PalominitosIsland bysite (mean+ 1 standarderror,n = 4). Sitesaredefined in Table 1, footnote b.
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120 100 _ 80 0-<i 60 40 20I 200 6 12 18 24 Time(hr) 70 I I I I I 60- 0VCSite C-sd - b*VCSit T-sed 50 - CM ECSiteC-sd _ O ECSiteTsed 40 20 10 -I I l l 0 6 12 24 Time(hr)
FIG. 7. Changes inpercentactivity for I coli(EC),asmeasured by AODC (A), andih measured by INT (B), for chambersinsed nitos Island by site (mean ± 1 standard e
defined in Table 1,footnote b.
Since mostof these same habitatsare harvesting areas, the resultsfurther er monitor contaminant levels of pathoge habitats.
ACKNOWLEDGMEN We aregrateful for the technical assista
Domingo, Luz Rodrfguez, Enid Elias, Frai
menOrtiz, Maria Guerra, Emilia Medina,C
Valdes-Collazo. GaryA.Toranzos maden
tothemanuscript. WeareindebtedtoIvet
useof herCoulter Counter.The PuertoRic( Resourcesand the Department of Marine
PuertoRico, Mayaguez,generously supplii ationfor these studies.
This workwassupportedby Sea Grant R partby the WaterResources Research Insti Puerto Rico at Mayaguez and by Public
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