Table Of ContentEFFECT OFENVIRONMENTAL SALINITYONTHEAPICAL
SURFACE OF CHLORIDE CELLS OFTHE EURYHALINETELEOST,
MUGILPLATANUS (PISCES,MUGILIDAE)
José Claro da Fonseca Neto '-^
Henry Louis Spach *
ABSTRACT
Juvenile mullets Mugil platanus (Günther, 1880) were transferred from saltwater to waters with
decreasingsalinitiesinordertoobservechangesinthegillepitheliummorphology,niainlyattheapicalsurface
ofChloridecells (CCs)oftheafferentfilamentside.TheopeningsofjuvenilesCCsadaptedto seawaterare
narrowanddeepamongthebordersofadjacentpavementcellswith =0.5- l|J.m. Changesinultrastructure
wereobservedwithin 15minafterthetransfertofreshwater.Atfirst,thesurfaceofopeningsbecamelarger,
usuallywith =0.5 - l|im, andexhibitedmicrovilli. Sinceopeningswithdifferentsizeswerepresentafter
6hinfreshwater,theresponseoftheoperingsofCCs inM.platanusadaptedtofreshwaterwasnotanall-or-
none phenomenon.
KEYWORDS. Gill arch, chloridecells, Mugilplatanus.
INTRODUCTION
Chloride cells (CCs) appear to be responsible for the osmotic regulation offish
adapted to sea water and freshwater due to their columnar position in the epithelia, the
high number of mitochondria in the cytoplasm, the basolateral membranes in close
association with the mitochondria and the presence of Na-K-ATPase in intimate
association withthe tubular System membrane (Pisam, 1981; Karnaky et al., 1984).
Another strong evidence for the participation of CCs in the osmoregulation
processes is the ultrastructural modification associated with the Na-K-ATPase enzyme,
that occurs when the fish adapt to environmental changes. During the adaptation of
euryhalinefishtoseawater,thefollowinghasbeenobserved: (1)anincreaseinenzymatic
activity (HossLERetal., 1979; Epsteinetal.,1980), (2) anincreaseinsizeandnumberof
CCsandmitochondria(Shirai&Utida, 1970;Pisam, 1981),(3)agreaterdevelopmentof
1. Centro de Estudos do Mar, UniversidadeFederal do Paraná . Av. BeiraMarS/N 83255-000, Pontal do
Paraná, Pontal do Sul, Paraná, Brasil, e-mail: [email protected]; hspach® aica.cem.ufpr.br.
Iheringia, Sér. Zool., Porto Alegre, (85): 151-156, 27 novembro 1998
152 FonsecaNeto& Spach
the tubular System (Shirai & Utida, 1970) and (4) a development of accessory CCs
forming "leaky junctions" (Pisam et ai., 1990). During adaptation to freshwater, the
processisinvertedandadecreaseinenzymeactivityoccurs,aswellasinthenumberand
sizeofCCsandmitochondria,inthetubularSystem (Shyrai&Utida, 1970;Evans, 1993)
and in general the intercellular complexe between the CCs and the accessory CCs
disappears.
SeveralstudieshavebeenperformedtoevaluatetheeffectonCCsofeuryhalinefish
in distinctenvironments. However, the majority ofthese studies consistedon adapting
freshwaterfishto seawaterandonlyfewhave attemptedtheopposite. This study, using
scanning electron microscopy (SEM), aims to show apical changes of CCs the gill
epitheliaof youngmuWetsMugilplatanus(Günter, 1880),whensubmitted todecreasing
salinity gradients.
MATERIALAND METHODS
YoungMugilplatanusof28to33mmwerecapturedduringthesummerof1995,usinga7.0mx2.0
mbeachseinenetand 1 mmwiremeshincreeiíssituatedonthebeachatPontaldoSul,Paraná,Brazil.They
weretransportedandmaintainedinacontrolledtemperaturaChamberat25°C,during7daysinfibertankswith
300Lofseawaterwiththesamesalinityofthecollectionárea. Duringthisperiod,fishwerefedonceaday
withpelletrations such aPiráTropical Growth in aproportionequivalentto20% ofthe tankbiomass.
Afterthe acclimation period, 20 individuais were transferred directly tobuckets containing 16 Lof
waterwithsalinitiesof34%c(control), 15%c,5%o,3%c, l%oand0%c(freshwater),includingareplicaforeach
salinity. Throughout the experiments the water was aerated and the fish were not fed. At the end ofeach
sampling,deadfishwereremoved,thedetritusweresiphonedandthewaterleveiwasreadjusted.Individuais
thatshowedsignsofstress,suchasadarkeneddorsum,in^egularswimming,orthosewhichremainedimmobile
on thebottomormovedonly when stimulatedmechanically, werenotusedforthechloride cell analysis.
Fromeachbückettwoindividuaiswerecollectedinatimeframeof 1, 3,6, 12, 24,48and96hafter
thebeginningoftheexperiment. IntheO%csalinity,observationsweremadeat 15, 30,45 minand 1, 3and
6h.Thefishweredecapitatedandthegillarcheswerecarefullyremovedfromtheopercularcavities. Inthe
34%cand15%osalinities,thearcheswererinsedwithaCurtlandSolution.Intheothersalinities,thearcheswere
rinsedwithasalineSolution(NaCl0.9%).Thesecondleftgillarchwasremovedandfixedfor24hat4-5°C
with3%glutaraldehydebufferedwith0.2M sodiumcacodilate(pH7.2),andthenrinsed.Theywereto50%
ethanolfor 15minandthenpreservedin75%ethanolatatemperature of4-5°C.InpreparationfortheSEM,
thenarchesweresuccessivelytransferredto90%ethanolandtwoseriesof100%ethanolandmaintainedfor
15 min in each concentration, dehydrated with CO, and sputter coated on an aluminum support. The
observations and electron micrographs were perfomed with Scanning Eletronic Microscopes Phillips (SEM
505)andJEOL(JSM840A).Theelectronmicrogaphswereclassifiedaccordingtosalinityandtimetreatments
andthe largestand smallestaxisoftheCC openings were measured. In ordertotestthehypothesis thatthe
openings do notvary accordingtotime and salinity, the t-testand two-way analysis ofvariance were used.
When significam differences were observed (p< 0.05), a test ofminimum significant difference was used
(SnEDECOR & COCHRAN, 1980).
The material is deposited in the reference collection ofthe Ichthyoplankton Laboratory , Centro de
EstudosdoMar, UniversidadeFederaldo Paraná, Pontal do Sul.
RESULTS
In ali the salinities used, many openings were randomly distributed among the
pavement cells, in the afferent side at the base of the secondary lamellae and in the
interlamelarregion ofthe gill filament epithelium (fig. 1).
The mean, standard deviation and range values ofthe large and small axis ofthe
chloridecellsareshownintableI.Alterationsinthesurfaceultrastructureoftheepithelial
Iheringia, Sér. Zool., PortoAlegre, (85): 151-156, 27 novembro 1998
Effect ofen\'ironniental salinit)' on the apical surface of.. 153
Figs. 1-6. GillfilamentsofMiigilplataniisexposedto34,3, 1 and0%salinities: I,afferentregionoffilament
showingCCopeningsin 34% salinityafter96h: 2, detailofCCopeningin 34% salinity after96h; 3,detail
ofCCopeningin3%salinityafter 1 h.Notethepresenceofmicrovilh;4,detailofCCopeningin 1%salinity
after 12h.Notethepresenceofmocrovilli;5,afferentregionoffilamentshowingCCopeningsin0%salinity
after30min; 6, detailofCC openingwithmany microvilliin0% salinity after 1 h. Bars l|im.
Iheringia, Sér. Zool., Porto Alegre, (85): 151-156, 27 novembro 1998
154 FonsecaNeto& Spach
Table 1.Range,meanandstandarddeviationofthelargeandsmallaxisofthechloridecellopeningsingills
ofjuvenileMugilplatanus in all salinities during 96h experiment.
Salinity Largeaxis (^m) Mean (|i,m) Smallaxis (|a.m) Mean (|xm)
34%o 0,50- 1,50 0,82 ± 0,24 0,50- 1,00 0,70±0,24
15%o 0,50- 1,50 0,95 ± 0,27 0,50- 1,50 0,79±0,26
5%o 0,50- 2,00 0,95 ±0,28 0,50- 1,00 0,77 ±0,24
3%o 0,30- 3,00 1,02±0,41 0,30 - 2,50 0,73 ±0,26
\%c 0,50- 3,50 1,15 ±0,52 0,30 - 2,50 0,87 ±0,37
Freshwater 0,50- 5,00 1,96±0,89 0,50-4,00 1,35 ±0,65
openings after 96 h in 34%o, 15%o and 5%o salinities were not significant (fig. 2).
Inthe 3%osalinity, theopenings showed some alterationinthe size andmicrovilli
appearafter Ihofexperiment(fig.3).Thefishin \%csalinityshowedlargeropeningsand
the microvilli were more frequent (fig. 4). In freshwater, after 15 min of experiment,
openings with increased dimensions and microvilli were clearly visible. (figs. 5,6).
A two-way analysis ofvariance in the larger axis showed significant differences
between salinities, time, and an interaction between these two factors. A multiple
comparisontestofminimumsignificantdifferences(MSD)showedthatthisaxisobtained
greatermeanlengths in 0%í>salinity, followedby l%o, bothbeinglargerthaninallother
salinities. Non-significantdifferences were foundbetween the mean values of3%o, 5%o
and 15%o salinities, and the mean values in the 5%o and 15%o salinities were not
significantly different from the 34%o salinity means. Regarding the mean length ofthe
largeraxisthroughoutthetimeframe,theMSDshowedasignificantlygreatermeanvalue
after6h ofexperimentation. Significantdifferences did not occurbetween the values of
the other time frames (tab. II).
TableII. Resultsoftwo-wayanalysisofvarianceappliedinthegreaterandlesseraxisoftheCCopenings in
Mugilplatanus,comparingsalinitiesandtimes. Resultsofsignificantdifferencesbymultiplecomparisonof
minimum significantdifferencestest (MSD). NS- non-significantdifference; *P<0.05.
LARGERAXIS SMALLERAXIS
F F
SALINITY (SAL) 3.65* 1.95NS
TIME (T) 3.28* 4.28*
INTERACTION 3.00* 2.45*
MSD LARGERAXIS SAL0%. SAL l%o SAL3%o SAL5%c SAL 15% SAL34%c
T6 Tl T3 T12 T24 T48 T96
Concemingthe smalleraxis, atwo-way analysis ofvariancepointedto significant
Iheringia, Sér. Zool.. PortoAlegre, (85): 151-156, 27 novembro 1998
Effect ofen\'ironmental salinity on the apical surface of... I55
differences between time and to the interaction between the effects ofsahnity andtime,
withnosignificantdifferencesbetweensalinities.Theuseofthemultiplecomparisontest
of minimum significant difference pointed to a greater mean length after 6 h of
experimentation.Moreovertheaveragesat 1,3and48haresimilartothoseat96h,which
are significantly greaterthan the means at 12 and 24h ofthe experiment (table II).
DISCUSSION
ThemorphologicalchangesofCCsareperhapsthebestdocumentedresponsesfor
thetransferofaeuryhalinefishfromfreshwatertoseawaterandviceversa.WhileKessel
&Beams(1962)andPhilpott&Copeland(1963)didnotobserveevidentchangesinCCs
when exposed to sea water, other authors noted an increase in their number and size
&
(Shirai Utida, 1970; Pisam, 1981; Pisam et al., 1987).
According to Maina (1990) the adaptation ofa fish to a new environment occurs
during a brief period, being faster in freshwater than in sea water. In juveniles of M.
platanus,therewasarapidresponsetothereductionofsalinitymainlyinfreshwaterwith
concentrationsunder5%,whereanotedincreaseoftheopeningsintheCCswasobserved
after only 15 min of experimentation. Rapid alterations were also observed in Mugil
cephalusLinnaeus, 1758(Hossler, 1980),inwhichchangesintheopeningsoccuredafter
3 h in freshwater. Slower responses were observed in Lebistes reticulatus Peters, 1859
(Straus, 1963) andAnguillajaponica Temmick & Schlegel, 1912 when transferred to
freshwater (Shirai & Utida, 1970). Such differences could be correlated with the
osmoregulatory ability ofthe fish (Hwang & Hirano, 1985).
In freshwater-adapted teleosts, the CCs showed openings with large diameters
containingmanymicrovilli(Philpott&Copeland, 1963;Hossler,1980).Thesealterations
ofthe CC openings were observed injuvenileM. platanus and were similarto changes
previously reported foM. cephalus by Hossler et al. (1979) and Hossler (1980).
Hossler (1980) observed that in M. cephalus, maintained in 10%o salinity, the
openings oftheCCs measuredfrom 1 to5 )imand showednumerous microvilli ontheir
surface.After24hinfreshwater,theopeningshadameandiameterof3-6|j,m.Considering
the differences among species and fish size, the changes observed in this study were
different from those observed by Hossler (1980) in M. cephalus. In M. platanus the
openings measured 0.50 x 1.50|im in sea waterand 0.50 x 5|j.m in freshwater.
Maina (1990) observed that the morphological modifications of the CCs in
OreochromisalcalicusgrahamiBoulenger, 1910arenotanall-Or-nonephenomenon,and
thatthedegree ofresponses is dependentonthe salinity gradientofthe environment, on
the species, and on the stage of development. The different leveis of morphological
modificationsofCCsfromthegillof M.platanussubmittedtothesametreatmentappear
to agree with the Statement that the CCs' responses are not uniform.
Whenthefisharetransferredtofreshwater,themorphologicalresponsesmaybethe
consequence of physiological response. Epstein et al. (1980) transferred Anguilla sp.
adaptedto seawater,tofreshwaterforonly2h, andobservedasuddendropinfluxrates
of Na""andCl",withouthteconcomitantalterationinNaK"ATPase.Thissuggeststhatthis
flux change could be the result of the effect of hormones (prolactin) on the "leaky
junctions" ofthe CCs-CCs or on the permeability ofthe apical membrane ofthe CCs,
providing an immediateprotection againstdemineralization. This immediate protection
Iheringia, Sér.*'ZooI., Porto Alegre, (85): 151-156, 27 novembro 1998
J56 FonsecaNeto& Spach
couldexplaintheabsenceofdeathsamongthejuvenilesofM.platanustransferredto 15%o
and 5%o salinities. Although morphological variations were present, they did not show
significantdifferencesfromthecontrolgroup. AccordingtoHwang&Hirano(1985)the
intermediarysalinitiesprovideonlypartoftheCCs'responses.Suchhormonalprocesses,
as well as the substantial and rapid alterations in CC openings, do not appear to be
sufficientforthesurvivalofthejuvenileM.platanustransferreddirectlyto3%o, \%cand
0%o salinities.
Acknowledgments. To CAPES, the Centro de Estudos do Mar-UFPR, the Centro de Microscopia
Electrônica-UFPRandtheLaboratóriodeMicroscopiaElectrónica, InstitutodeFísica, UniversidadedeSão
Paulo, fortheircollaboration in this study.
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