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Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp
Macrobrachium amazonicum (Decapoda, Palaemonidae)
N.M. Belli b, R.O. Faleiros b, K.C.S. Firmino a, D.C. Masui a, F.A. Leone a, J.C. McNamara b, R.P.M. Furriel a,⁎
a Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, São Paulo, Brazil
b Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, São Paulo, Brazil
a b s t r a c ta r t i c l e i n f o
Article history:
Received 22 October 2008
Received in revised form 19 November 2008
Accepted 20 November 2008
Available online 6 December 2008
Keywords:
Gill Na,K-ATPase kinetics
K-phosphatase activity
α-subunit expression
Gill ultrastructure
Gill epithelial interfaces
Macrobrachium amazonicum
Salinity acclimation
Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life
cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to
survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics
and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-
days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na+ uptake across the gill
epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit
expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes
redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking
between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an
intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between
the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal
cell invaginations, respectively. These findings are discussed regarding the putative movement of Na+ across
the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in
their invaginations.
© 2008 Elsevier Inc. All rights reserved.
1. Introduction
The evolutionary history of the aquatic Crustacea reveals broad
radiation from the ancestral marine environment into estuaries, rivers
and lakes, and the invasion of freshwater habitats by the many
different successful crustacean taxa is underpinned by behavioral,
reproductive, morphological, physiological and biochemical adapta-
tions. Their notable ability to maintain the solute concentration of the
extracellular fluid well above that of the external medium is
particularly significant and derives in part from decreased body
surface permeability to water and ions (McNamara et al., 2005) and
enhanced ion uptake via transporters located in the gill epithelia
(Péqueux, 1995; Lucu and Towle, 2003; Freire et al., 2008).
A central role for the gill Na,K-ATPase in active Na+ uptake into the
hemolymph of hyperosmoregulating crabs has been well established
(Péqueux,1995; Lucu and Towle, 2003; Kirschner, 2004). An apical Na+
/H+ exchanger and a Na+/K+/2Cl− co-transporter apparently mediate
Na+ flux into the gill cells of weak hyper-regulators while an apical V-
ATPase and Na+ channels may prevail in strong hyper-regulators
(Onken and Riestenpatt, 1998; Onken et al., 2003; Kirschner, 2004).
The basally located Na,K-ATPase then exports 3Na+ from the cytosol to
the hemolymph, concomitantly importing 2K+ at the expense of ATP
hydrolysis. Little information is available for hyper-regulating car-
idean shrimps.
There is a clear link between hyper-regulation and gill Na,K-ATPase
activity, which decreases several-fold when crustaceans from fresh
water or dilute media are acclimated to elevated salinities (reviewed
by Péqueux, 1995; Lucu and Towle, 2003; Leone et al., 2005a; Freire
et al., 2008). For example, Na,K-ATPase specific activity is elevated in
gills of the freshwater shrimps Macrobrachium amazonicum (Heller)
and M. olfersi in fresh water (Furriel et al., 2000, 2001; Santos et al.,
2007) and declines about 2-fold on acclimation of M. olfersi to 21‰
salinity (Mendonça et al., 2007). The molecular mechanisms under-
lying such long-termmodulation of Na,K-ATPase activity are the focus
of considerable interest. Transcriptional and post-transcriptional
mechanisms may be involved, and include changes in transcription
rate and/or mRNA stability, changes in translation and protein
degradation rates, differential RNA splicing or post-translational
modifications, and trafficking of enzyme molecules between the
plasma membrane and intracellular pools (Towle et al., 2001; Henry
et al., 2002; Lucu and Towle, 2003; Masui et al., 2005; Luquet et al.,
2005; Lovett et al., 2006a; Chung and Lin, 2006; Jayasundara et al.,
2007). The expression of different isoenzymes also may be pertinent,
as suggested for some crabs (Siebers et al., 1983; Harris and Bayliss,
Comparative Biochemistry and Physiology, Part A 152 (2009) 431–439
⁎ Corresponding author. Tel.: +55 16 3602 3749; fax: +55 16 3602 4838.
E-mail address: rosapmfi@ffclrp.usp.br (R.P.M. Furriel).
1095-6433/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2008.11.017
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1988; Genovese et al., 2004; Masui et al., 2005; Lovett et al., 2006a)
and M. olfersi (Mendonça et al., 2007). Higher specific activities are
associated with the differentiation and hypertrophy of ion-transport-
ing cells in the gill epithelia of hyper-regulating crabs and lobsters
exposed to dilute media (Lucu and Towle, 2003; Lovett et al., 2006b).
The Na,K-ATPase, a member of the P2C subfamily of P-type ATPases,
consists of a 110-kDa catalytic α-subunit, and a highly glycosylated
≈50-kDa β-subunit, necessary for the maturation, targeting, stability
and functional expression of the enzyme. Like all P-type ATPases, the
Na,K-ATPase α-subunit is reversibly phosphorylated at a conserved
aspartate residue during its reaction cycle, and both the phosphory-
lated and unphosphorylated forms undergo conformational transi-
tions (E1→E2 and E1P→E2P), essential for cation transport (reviewed
by Horisberger, 2004; Martin, 2005). The enzyme also hydrolyzes
other phosphate-containing substrates like p-nitrophenyl phosphate
(pNPP) (Drapeau and Blostein, 1980; Robinson, 1981, 1985; Robinson
et al., 1983; Glynn, 1985; Gatto et al., 2007) and, although the kinetic
mechanism of pNPP hydrolysis is unclear, the E2 form seems to be the
main conformation involved (Glynn,1985; Robinson and Pratap,1993;
Jorgensen et al., 1998). Further, there is a strict correlation between the
ouabain-sensitive ATPase and K-phosphatase activities, since these
correspond to different activities of the same enzyme (Robinson,1981;
Glynn, 1985; Esmann, 1988; Furriel et al., 2001; Masui et al., 2003).
The diadromous cinnamon shrimp, M.amazonicum (Heller), is
endemic to the Orinoco, Amazon, and Paraguay River basins, but is
also found in north/eastern Brazil and the upper Paraná River owing to
anthropic activity (Ramos-Porto and Coelho, 1998; Bialetzki et al.,
1997; Magalhães et al., 2005). Different populations occupy habitats
ranging from fresh to estuarine waters and differ in osmoregulatory
ability and salt-dependent larval survival (McNamara et al., 1983;
Zanders and Rodriguez, 1992; Collart and Rabelo, 1996). Augusto et al.
(2007) have examined isosmotic intracellular and anisosmotic
extracellular regulatory ability in a land-locked population of M.
amazonicum from southeastern Brazil, subject of the present study.
Members of this population maintain strong hemolymph osmotic and
ionic gradients in freshwater, becoming isosmotic and iso-ionic after
5 days at 22‰ salinity. The gill ultrastructure of M. olfersi (Freire and
McNamara, 1995) and M. amazonicum (Freire et al., 2008) has been
investigated and putative routes of salt movement into the hemo-
lymph proposed. In such freshwater shrimps, the gill epithelium
typically consists of structurally linked pillar cells and mitochondria-
rich, intralamellar septal cells, which exhibit membrane surfaces
highly amplified by apical microvilli and deep invaginations, respec-
tively (McNamara and Lima, 1997). The septal cell membrane system
houses the Na,K-ATPase (McNamara and Torres, 1999) and is bathed
by the hemolymph as are the pillar cell bodies; the apical pillar cell
surfaces below the cuticle face the external medium.
To investigate the suite of alterations transpiring during the
reproductive migration of palaemonid shrimps into saline media, in
this study we provide a kinetic characterization of the K-phospha-
tase activity of the Na,K-ATPase in gill microsomes from M.
amazonicum held in fresh water or acclimated to 21‰ salinity for
10 days. We also examine the membrane interfaces between the
pillar and intralamellar septal cells and their respective media, in
shrimps in fresh water, to better characterize the likely locations of
ion transporters and potential pathways of ion movement. We
demonstrate a substantial decrease in K-phosphatase activity,
lowered α-subunit protein levels, alterations in protein expression
pattern and a redistribution of enzyme activity into membrane
fractions of different densities in salinity-acclimated shrimps. The
membrane interfaces between the external medium and the
hemolymph are greatly augmented by apical pillar cell evaginations
and septal cell invaginations, respectively, which house the
transporter and active ion pump systems. These findings are
discussed as regards the uphill movement of Na+ across the pillar-
septal cell boundaries into the hemolymph.
2. Materials and methods
2.1. Materials
All solutions were prepared using Millipore MilliQ ultrapure
apyrogenic water, and all reagents were of the highest purity
commercially available. Imidazole, Hepes, ouabain, pNPP, ethacrynic
acid, oligomycin, thapsigargin, bafilomycin A1, sodium orthovanadate,
nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indole (BCIP), aur-
overtin B, molecular weight marker mixture, alamethicin, parafor-
maldehye, glutaraldehyde, sodium cacodylate and osmium tetroxide
were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).
Dimethyl sulfoxide was from Merck. The protease inhibitor cocktail
(5 mmol L−1 leupeptin, 5 mmol L−1 antipain, 1 mmol L−1 benzamidine
and 1 mmol L−1 pepstatin A) was from Calbiochem. The α-5
monoclonal antibody against the α-subunit of avian Na,K-ATPase
(all isoforms) was purchased from Developmental Studies Hybridoma
Bank (University of Iowa, IA, USA). Antimouse IgG, alkaline phospha-
tase conjugate was purchased from Promega Corporation (Madison,
WI, USA). Sodium orthovanadate solution was prepared according to
Furriel et al. (2000).
2.2. Study material and salinity acclimation
Adult, intermolt M. amazonicum, approximately 10–15 cm long,
were collected using a builder's sieve from the marginal vegetation of
the Usina Santa Elisa reservoir (salinity b0.5‰, ≈23 °C), Sertãozinho
(21° 06′ 47″ S; 48° 03′ 18″W) in northeastern São Paulo State, Brazil. In
the laboratory, the shrimps were acclimated for 10 days either to
reservoir water or to dilute seawater (21‰ salinity, 630 mOsm/kg
H2O) under a natural light/dark photoperiod at ≈25 °C. Dilute seawater
was prepared from Maresias Beach seawater (33.5‰ salinity) and
reservoir water; salinity was verified using a handheld refractometer
(American Optical, Model 10419, Buffalo, NY, USA). Shrimps were fed
on alternate days with minced bovine muscle.
2.3. Preparation of gill microsomal fractions
Biochemical analyses were performed on gill microsomes pre-
pared from shrimps held in fresh water or acclimated to 21‰ salinity
for 10 days. After briefly chilling the shrimps in crushed ice, all gill
pairs were rapidly dissected on ice in homogenization buffer
(20 mmol L−1 imidazole, pH 6.8, containing 6 mmol L−1 EDTA,
250 mmol L−1 sucrose). For each homogenate, the gills from 15–20
shrimps were diced and homogenized in homogenization buffer
containing the protease inhibitor cocktail (20 mL buffer/g wet tissue),
using a Potter homogenizer. After centrifuging the crude extract at
10,000 ×g for 35 min at 4 °C, the supernatant was placed on crushed
ice; the pellet was re-homogenized as above, and re-centrifuged
under the same conditions. The two supernatants were then pooled
and centrifuged at 100,000 ×g for 2 h at 4 °C. The resulting pellet was
homogenized in 20 mmol L−1 imidazole buffer, pH 6.8, containing
250 mmol L−1 sucrose (15 mL buffer/g wet tissue). Finally, 0.5 mL
aliquotswere rapidly frozen in liquid nitrogen and stored at −20 °C. No
appreciable loss of microsomal activity was seen after a two-month
storage, either in shrimps maintained in fresh water or those
acclimated to 21‰ salinity. When required, the aliquots were thawed,
placed on crushed ice and used immediately.
2.4. Measurement of pNPP hydrolysis
pNPP hydrolysis by gill microsomal fractions was assayed con-
tinuously at 25 °C, monitoring the release of the p-nitrophenolate ion
(ε410 nm, pH 7.5=13,160 mol−1 L cm−1) in a FEMTO 700 plus spectro-
photometer equipped with thermostatted cell holders. Standard assay
conditionswere: 50mmol L−1 Hepes buffer, pH 7.5, containing 10mmol
432 N.M. Belli et al. / Comparative Biochemistry and Physiology, Part A 152 (2009) 431–439
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L−1 pNPP, 10 mmol L−1 KCl and 10 mmol L−1 MgCl2 (shrimps held in
fresh water); or 8 mmol L−1 pNPP, 10 mmol L−1 KCl and 5 mmol L−1
MgCl2 (shrimps acclimated to 21‰ salinity), in a final volume of 1.0 mL.
Controls without added enzyme were included in each experiment to
quantify the non-enzymatic hydrolysis of substrate.
When using microsomal preparations from salinity-acclimated
shrimps, pNPP hydrolysis was also estimated in the presence of
3 mmol L−1 ouabain to assess ouabain-insensitive activity, the
difference in measured pNPPase activity in the absence or presence
of ouabain representing the K-phosphatase activity. For microsomal
preparations from shrimps maintained in fresh water, ouabain-
insensitive activity was negligible and total pNPPase activity corre-
sponded to the K-phosphatase activity. pNPPase activity was also
assayed in the presence of alamethicin (0.5 to 3.0 μg/μg protein) to
evaluate the presence of sealed vesicles in the microsomal prepara-
tions, as described by Furriel et al. (2000). Initial velocities were
constant for at least 15min, provided that less than 5% of the substrate
was hydrolyzed. Assays were performed in duplicate aliquots and each
experiment was repeated using three different gill homogenates
(N=3). One enzyme unit (U) is defined as the amount of enzyme that
hydrolyzes 1.0 nmol of pNPP per min, at 25 °C.
2.5. Continuous-density sucrose gradient centrifugation
An aliquot of the Na,K-ATPase-rich microsomal fraction was
layered into a 10 to 50% (w/w) continuous-density, sucrose gradient
in 20mmolL−1 imidazole buffer, pH 6.8, and centrifuged at 180,000 ×g
and 4 °C for 2 h using a PV50T2 Hitachi vertical rotor. Fractions
(0.5 mL) collected from the bottom of the gradient were then assayed
for total pNPPase activity, ouabain-insensitive pNPPase activity,
protein, and refractive index.
2.6. Western blot analysis
SDS-PAGE and Western blot analyses of the gill microsomes from
shrimps held in fresh water or acclimated to 21‰ salinity were
performed as described by Furriel et al. (2000). Briefly, after
electrophoretic separation of the proteins, the gel was split: one half
was stained with silver nitrate and the other electroblotted using a
Gibco BRL Mini-V 8-10 system, employing a nitrocellulose membrane
that was then incubated for 1 h at 25 °C in a 1:10 dilution of the α-5
monoclonal antibody. After washing three times in 50 mmol L−1 Tris–
HCl buffer, pH 8.0, containing 150 mmol L−1 NaCl and 0.1% Tween 20,
the membrane was incubated for 1 h at 25 °C with an anti-mouse IgG,
alkaline phosphatase conjugate, diluted 1:7500. Incorporation of the
specific antibody was developed in 100 mmol L−1 Tris–HCl buffer, pH
9.5, containing 100 mmol L−1 NaCl, 5 mmol L−1 MgCl2, 0.2 mmol L−1
NBT and 0.8 mmol L−1 BCIP. Controls consisting of membranes
incubated with the secondary antibody without previous incubation
with the α-5 antibody were included in each experiment. Immuno-
blots were scanned and imported as JPG files into a commercial
software package (Kodak 1D 3.6) where the immuno-reaction
densities were quantified and compared. Western blot analysis for
each experiment was repeated 3 times using different tissue
preparations from separate pools of 15–20 shrimps each.
2.7. Measurement of protein
Protein concentration was estimated according to Read and
Northcote (1981) using bovine serum albumin as the standard.
2.8. Estimation of kinetic parameters
The kinetic parameters VM (maximum velocity), K0.5 (apparent
dissociation constant), KM (Michaelis–Menten constant) and the nH
value (Hill coefficient) for pNPP hydrolysis were calculated using
SigrafW software (Leone et al., 2005b). The curves presented are those
that best fit the experimental data. The kinetic parameters provided in
the tables are calculated values and represent the mean±SD derived
from three different microsomal preparations (N=3). The apparent
dissociation constant of the enzyme-inhibitor complex, KI, was
estimated as described by Furriel et al. (2000).
2.9. Ultrastructural analysis
Gill ultrastructure was examined in shrimps kept in fresh water.
Palaemonid gills are not differentiated into anterior and posterior
types as found in crabs (Freire et al., 2008), although the sixth gill is
the largest. After chilling the shrimps briefly on ice, both branchios-
tegites were removed and the sixth gill pair was dissected under
primary fixative on ice. Medial gill portions, comprising about 10
lamellae each, were excised from each gill and placed in primary
fixative on ice for 1.5 h. The primary fixative, containing cation
concentrations specifically adjusted to M. amazonicum hemolymph
osmolality and composition (Augusto et al., 2007), consisted of
100 mmol L− 1 sodium cacodylate buffer, pH 7.4, containing
250 mmol L−1 glutaraldehyde, 200 mmol L−1 paraformaldehyde,
65 mmol L−1 NaCl, 5 mmol L−1 KCl, 5 mmol L−1 CaCl2 and 1 mmol L−1
MgCl2 (estimated effective osmolality ≈450 mOsm/kg H2O). The
portions were then rinsed (3× 5 min) in buffer solution alone
(composition as above less aldehydes, ≈360 mOsm/kg H2O) and
post-fixed in 1% osmium tetroxide in the same buffer for 1.5 h on ice.
Dehydration, embedding in Araldite 502 epoxy resin and sectioning
followed routine procedures (McNamara and Torres, 1999). Thin (50–
80 nm) sections, prepared using a diamond knife, were stained with
aqueous uranyl acetate and Reynolds' (1963) lead citrate and examined at
an accelerating voltage of 100 kV in a Philips EM208 electronmicroscope.
3. Results
3.1. Sucrose density gradient centrifugation
Sucrose density gradient centrifugation of the gill microsomal
fraction from M. amazonicum acclimated to 21‰ salinity showed two
Fig. 1. Sucrose density gradient centrifugation of a microsomal fraction from the gill
tissue of M. amazonicum acclimated to 21‰ salinity for 10 days. An aliquot containing
2 mg protein was layered into a 10 to 50% (w/w), continuous, sucrose density gradient.
Fractions (0.5 mL) were collected from the bottom of the gradient and analyzed for total
pNPPase activity (○), K-phosphatase activity (●), ouabain-insensitive pNPPase activity
(□), protein concentration (■) and sucrose concentration (Δ). Note that both peaks
exhibit similar ratios between total and ouabain-insensitive pNPPase activities. Inset:
Sucrose density gradient centrifugation of a microsomal fraction (905 μg protein) from
the gill tissue ofM. amazonicum held in fresh water (b0.5‰ salinity). Experiments were
performed using duplicate aliquots from three (N=3) different gill homogenates
prepared from 15–20 shrimps in each condition; representative curves obtained for one
homogenate in each condition are given.
433N.M. Belli et al. / Comparative Biochemistry and Physiology, Part A 152 (2009) 431–439
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well-defined peaks of K-phosphatase activity over the 25–30% and
35–42% sucrose ranges, coincident with protein peaks (Fig. 1). This
suggests that the Na,K-ATPase is distributed in membrane fractions of
different densities. Most of the activity was associated with lower
density membrane fragments, and both peaks exhibited ouabain-
insensitive pNPPase activities, indicating the presence of pNPP-
hydrolyzing enzymes other than the Na,K-ATPase.
Analysis of the microsomal fraction from shrimps held in fresh
water also revealed two protein peaks (inset to Fig. 1), the lighter (15–
20% sucrose) constituted by membrane fragments completely lacking
pNPPase activity. Heavier fractions (25–35% sucrose) exhibited K-
phosphatase but not ouabain-insensitive activity, indicating the
absence of phosphohydrolyzing enzymes other than the Na,K-ATPase.
K-phosphatase activity was negligible in fractions from the 35–42%
sucrose range.
3.2. SDS-PAGE and Western blot analysis
SDS-PAGE and Western blot analyses of gill microsomes from
shrimps held in fresh water (lanes A and C) or acclimated for 10 days
to 21‰ salinity (lanes B and D), employing equal amounts of protein
per slot, are shown in Fig. 2. The electrophoretic profiles (lanes A and
B) are clearly different, suggesting altered protein expression in
response to salinity acclimation. Western blot analysis using the α-5
monoclonal antibody against the α-subunit of avian Na,K-ATPase (all
isoforms) identified a single immunoreactive band of about 115 kDa in
both preparations. Quantitative image analysis revealed a 2.5-fold
higher density for the Na,K-ATPase α-subunit band in the microsomal
fraction from shrimps held in fresh water, indicating a substantial
decrease in the relative abundance of the enzyme in response to
salinity acclimation.
3.3. Kinetic characterization of K-phosphatase activity
Stimulation of K-phosphatase activity in gill microsomes from M.
amazonicum maintained in fresh water or acclimated to 21‰ salinity
Fig. 2. SDS-PAGE and Western blot analyses of microsomal fractions from the gill tissue
of M. amazonicum acclimated to 21‰ salinity for 10 days or held in fresh water.
Electrophoresis was performed in a 5–20% polyacrylamide gel using 4 μg microsomal
protein for silver staining and 80 μg for Western blotting. Silver nitrate-stained gels:
lane A — shrimps in fresh water; lane B — shrimps acclimated to 21‰ salinity. Western
blotting against Na,K-ATPase α-subunit: lane C — shrimps in fresh water; lane D —
shrimps acclimated to 21‰ salinity. The analysis was repeated three times using
aliquots from different gill homogenates prepared from shrimps in each condition; a
representative figure is shown. Controls consisting of membranes incubated with the
secondary antibody alone were included in each experiment;no bands were revealed
(not shown).
Fig. 3. Modulation of K-phosphatase activity by pNPP, and magnesium, potassium and ammonium ions in microsomal fractions from the gill tissue of M. amazonicum acclimated to
21‰ salinity for 10 days or held in fresh water. Activity was assayed continuously at 25 °C in 50 mmol L−1 Hepes buffer, pH 7.5, in a final volume of 1.0 mL. The modulation of enzyme
activity by each effector was evaluated under optimal concentrations of the others. A— pNPP; B—Magnesium ions; C— Potassium ions; D— Ammonium ions. Inset to Fig. C: effect of
K+ concentration on K-phosphatase activity in the presence of saturating NH4+ concentrations. Inset to Fig. D: effect of NH4+ concentration on K-phosphatase activity in the presence of
saturating K+ concentrations. (○) Shrimps in fresh water; (●) shrimps acclimated to 21‰ salinity. Experiments were performed using duplicate aliquots from three different gill
homogenates, prepared from shrimps in each condition; representative curves obtained for one homogenate in each condition are given.
434 N.M. Belli et al. / Comparative Biochemistry and Physiology, Part A 152 (2009) 431–439
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by pNPP, Mg2+ and K+, each under saturating concentrations of the
others, is provided in Fig. 3. In both cases, K-phosphatase activity was
stimulated by pNPP (Fig. 3A) following simple saturation curves,
obeying cooperative kinetics (nH=1.2). Although substrate K0.5 values
were similar (1.24±0.06 mmol L−1 and 1.16±0.07 mmol L−1 for
freshwater- and salinity-acclimated animals, respectively), VM was
2.5-fold higher in the freshwater-held shrimps (156.1±7.8 U mg−1),
congruent with the 2.5-fold higher relative abundance of Na,K-ATPase
in their microsomal fraction. pNPPase activity assays in the presence
of increasing alamethicin concentrations (0.5 to 3.0 μg/μg protein)
revealed the presence of sealed vesicles only in the microsomes from
shrimps acclimated to 21‰ salinity (data not shown). All activity
assays were thus performed using 2.0 μg alamethicin/μg protein, a
condition providing maximal pNPPase activity, probably owing to the
free access of substrate and ions to their sites on the enzyme
molecules.
The stimulation of K-phosphatase activity by Mg2+ (K0.5=1.03±
0.06 mmol L−1 and 1.38±0.09 mmol L−1 for freshwater- and salinity-
acclimated shrimps, respectively) and K+ (K0.5=2.36±0.14 mmol L−1
and 2.45±0.18 mmol L−1 for freshwater- and salinity-acclimated
shrimps, respectively) revealed site–site interactions, following single
titration curves (Fig. 3B and C). In the absence of K+ and presence of
saturating pNPP and Mg2+ concentrations, NH4+ also stimulated K-
phosphatase activity, revealing positive cooperativity (nH=2.2) up to
specific activities similar to those for K+ in the absence of NH4+ (Fig. 3D).
K0.5 values for the NH4+ stimulation of K-phosphatase activity were
similar in both acclimation conditions (11.4±0.7 mmol L−1 and 11.9±
0.5 mmol L−1 for freshwater- and salinity-acclimated shrimps,
respectively), and 4.8-fold higher than those for K+. NH4+ and K+ did
not synergistically stimulate K-phosphatase activity (insets to Fig. 3C
and D). The kinetic parameters calculated for the stimulation of K-
phosphatase activity by pNPP, Mg2+, K+ and NH4+ are summarized in
Table 1.
The effect of ouabain on total pNPPase activity in gill microsomal
fractions fromM. amazonicummaintained in freshwater or acclimated
to 21‰ salinity is shown in Fig. 4. Ouabain concentrations around
3 mmol L−1 completely inhibited activity in microsomes from shrimps
held in fresh water. However, a residual activity of about 11% was
estimated for salinity-acclimated shrimps, even at ouabain concentra-
tions as high as 7 mmol L−1. This finding indicates the presence of
pNPP-hydrolyzing enzymes other than the Na,K-ATPase only in the
microsomal fractions of shrimps acclimated to 21‰ salinity, in
agreement with the sucrose density gradient analysis (Fig. 1). The
monophasic inhibition curves obtained suggest the presence of a
single α-subunit isoform in the microsomal fractions, in accordance
with the Western blot data (Fig. 2). The calculated apparent
dissociation constants (KI) for ouabain were 176.1±8.8 µmol L−1 and
95.64±5.7 µmol L−1 for freshwater- and salinity-acclimated shrimps,
respectively (inset to Fig. 4).
To identify the enzymes responsible for the ouabain-insensitive
pNPPase activity in the microsomal fractions from M. amazonicum
acclimated to 21‰ salinity, we evaluated the effect of various
inhibitors together with 3 mmol L−1 ouabain on total pNPPase activity
(Table 2). Under saturating concentrations of pNPP, Mg2+, and K+, the
residual activity in the presence of ouabain alone (control) reveals that
the Na,K-ATPase accounts for around 90% of total activity. While the
95% inhibition of total activity by orthovanadate plus ouabain suggests
the presence of other P-type ATPases or neutral phosphatases in the
preparation, the lack of effect of thapsigargin and theophylline
suggests the absence of both Ca2+-ATPases and phosphatases. In
contrast, ethacrynic acid partially inhibited ouabain-insensitive
pNPPase activity, suggesting the presence of Na- and/or K-ATPases
in the preparation that may be sensitive to orthovanadate inhibition.
The absence of effect of bafilomycin A1 suggests that the preparation is
free of V-ATPase activity, while the partial inhibition of ouabain-
Table 1
Kinetic parameters for the stimulation by K+, Mg2+, NH4+ and pNPP and ouabain
inhibition of K-phosphatase activity in microsomal fractions from gills of M.
amazonicum held in fresh water (FW, b0.5‰ salinity) or acclimated to 21‰ salinity
(21‰) for 10 days
Effector V (U mg−1) K0.5 (mmol L−1) nH KI (µmol L−1)
FW 21‰ FW 21‰ FW 21‰ FW 21‰
PNpp 156.1±7.8 62.3±3.7 1.2±0.1 1.2±0.1 1.2 1.2 – –
Mg2+ 152.7±9.5 62.8±4.4 1.0±0.1 1.4±0.1 2.3 2.3 – –
K+ 149.6±9.0 57.9±4.2 2.4±0.1 2.5±0.2 1.2 2.0 – –
NH4+ 141.4±8.7 65.1±2.9 11.4±0.7 11.9±0.5 2.2 2.2 – –
Ouabain – – – – – – 176.1±8.8 95.6±5.7
Assays were performed in 50 mmol L−1 Hepes buffer, pH 7.5, in a final volume of 1.0 mL.
The response to each effector was evaluated under optimal concentrations of the others.
Data are the mean±SD from three (N=3) different microsomal fraction preparations.
Fig. 4. Effect of ouabain on total pNPPase activity of microsomal fractions from the gill
tissue of M. amazonicum acclimated to 21‰ salinity for 10 days or held in fresh water.
Activity was assayed continuously at 25 °C in 50 mmol L−1 Hepes buffer, pH 7.5, under
optimal conditions of substrate and ions. Inset: Dixon plots for estimation of KI, inwhich
vc is the reaction rate corresponding to K-phosphatase activity alone. (○) Shrimps in
fresh water; (●) shrimps acclimated to 21‰ salinity. The experiments were performed
using duplicate aliquots from three different gill homogenates prepared from shrimps
in each condition; representative curves obtained for one homogenate in each condition
are given.
Table 2
Effect of various inhibitors on pNPPase activitya of gill microsomes fromM. amazonicum
acclimated to 21‰ salinity for 10 days
% V U mg−1
Controls
No inhibitor 100.0 65.5±3.9
Ouabain (3.0 mmol L−1) 10.8 7.1±0.4
Orthovanadate (1.0 μmol L−1) 5.5 3.6±0.2
Ouabain (3.0 mmol L−1) plus
Orthovanadate (1.0 μmol L−1) 5.5 *3.6±0.2
Ethacrynic acid (2.0 mmol L−1) 6.4 *4.2±0.3
Aurovertin Bb (10.0 μmol L−1) 7.3 *4.8±0.3
Sodium Azide (0.1 mmol L−1) 4.6 *3.0±0.2
Tapsigarginc (0.5 μmol L−1) 11.8 7.7±0.5
EGTA (1 mmol L−1) 9.2 *6.0±0.4
Bafilomycin A1c (0.4 μmol L−1) 11.9 7.8±0.6
Theophylline (5 mmol L−1) 10.0 6.6±0.5
aAssays were performed continuously at 25 °C in 50 mmol L−1 Hepes buffer, pH 7.5,
containing 8 mmol L−1 pNPP, 5 mmol L−1 MgCl2, 10 mmol L−1 KCl, and 2 μg alamethicin/
μg protein, in a final volume of 1.0mL. Data are themean±SD from three (N=3) different
microsomal fraction preparations; bin ethanol; cin dimethylsulfoxide. Data were
analyzed using the Kruskal–Wallis one-way ANOVA on ranks (H=31.1, df=10,
Pb0.001) followed by Student–Newman–Keuls multipleranks testing (*P≤0.05
compared to value for 3.0 mmol L−1 ouabain).
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insensitive activity by aurovertin B and azide indicates the presence of
an F-type ATPase, possibly owing to contamination by mitochondrial
membranes.
3.4. Ultrastructural analysis of the gill epithelium
M. amazonicum possesses seven pairs of pleurobranchiae located in
the cephalothoracic gill chambers and arrayed as a double series of
flattened, phyllobranchiate hemi-lamellae that project from a trian-
gular base. The gill epithelium below the fine cuticle consists of squat
pillar cells disposed on both sides of the lamellae. The pillar cells
exhibit thick (2.5 µm), apical flanges, which extend radially below the
cuticle, making contact with adjacent flanges via junctional com-
plexes. Their perikarya abut basally on a simple layer of intra-lamellar
septal cells, forming a system of irregularly compartmented lacunae
through which hemolymph flows divided vertically by the pillar cell
bodies and horizontally by the septal cells (Fig. 5).
The boundary between the apical pillar cell membrane and the
cuticle consists of numerous, short (0.5±0.1 μm), fine (95 nm), semi-
regular evaginations and leaflets, each attached to the endocuticle by a
small electron-dense macula. A distinct although discrete electron-
lucent, subcuticular space extends for up to 1 μm into the electron-
dense apical cytoplasm that contains numerous small vesicles,
polyribosomes, rough endoplasmic reticulum cisternae and abundant
mitochondria (Fig. 6). The interface between the lower apical flange
surface and the hemolymph is simple and not folded or amplified,
consisting of the basal flange membrane itself underlain by the basal
lamina (Fig. 6). The junctions between adjacent pillar cell flanges
consist of a small desmosome-like structure followed by a septate
junction and a lengthy region of close apposition (20 nm) between the
juxtaposed cell membranes, leading to the hemolymph space (Fig. 6).
The intralamellar septum is extensive and unfenestrated, consist-
ing of septal cells characterized by abundant mitochondria sur-
rounded by numerous, deep invaginations of the plasmalemma
(Fig. 5). The interface between the septal cells and the hemolymph
is highly amplified, consisting of lengthy (≈4 μm), narrow (40 nm)
invaginations that extend profoundly into the septal cell cytoplasm,
each associated longitudinally with one or more mitochondria closely
apposed to the cell membrane on either side of each invagination,
forming a clearly structured unit (Fig. 7). The two membranes
comprising the invaginations in such regions appear to be thickened
and possess adhering electron-dense material (Fig. 7). The interface
between the pillar cell bases and the intralamellar septal cells ranges
Fig. 5. Gill epithelium ultrastructure in the freshwater shrimp, M. amazonicum.
Transverse section orthogonal to the long axis of a hemi-lamella from the sixth gill of
a shrimp held in fresh water (b0.5‰ salinity), revealing the typical palaemonid
epithelial architecture. The epithelium below the thin cuticle (c) consists of the robust,
apical pillar cell flanges (f) that extend radially above the stout pillar cell body (b)
containing the nucleus. Junctional complexes (arrows) connect adjacent flanges. The
unfenestrated intralamellar septum (s), consisting of highly invaginated septal cells,
forms two symmetrical compartments through which the hemolymph (h) flows,
broken at semi-regular intervals by the abutting pillar cell bodies. Scale bar=3 µm.
Fig. 6. Gill epithelium ultrastructure in the freshwater shrimp, M. amazonicum.
Transverse section of a hemi-lamella long axis, revealing the complex interface
between the apical pillar cell flange and the cuticle (c) that faces the external medium.
The apical membrane surface is thrown into an extensive system of cytoplasmic
evaginations (e) that form an ample subcuticular space (⁎) below which lie abundant
mitochondria, cisternae of the ample rough endoplasmic reticulum (r), free and
polyribosomes and coated vesicles. Adjacent pillar cell flanges are intimately coupled by
a small desmosome-like structure (d) followed by a septate junction (s) and a long
region of close membrane apposition (a) that leads to the hemolymph space (h). The
basal flange membrane of the pillar cell is not folded and is underlain by a finely
filamentous basal lamina (b). Scale bar=0.5 µm.
Fig. 7. Gill epithelium ultrastructure in the freshwater shrimp, M. amazonicum.
Transverse section through the mitochondria-rich intralamellar septum (s) in a region
showing simple juxtaposition (arrows) with the membrane of an abutting pillar cell
base (b) containing the nucleus (n) and characteristic RER cisternae (r). The septal cell
membrane is thrown into an extensive system of deep invaginations (i) each of which is
intimately coupled with numerous mitochondria (m), frequently forming areas of
structured apposition (⁎) characterized by a narrow cytoplasmic and extracellular space
of constant width (25 nm each) lying between adjacent mitochondria. The Na,K-ATPase
resides in these membranes (McNamara and Torres, 1999) and actively transports Na+
into the invagination lumens fromwhere the Na+-rich fluid moves into the hemolymph
by bulk flow. Scale bar=1 µm.
436 N.M. Belli et al. / Comparative Biochemistry and Physiology, Part A 152 (2009) 431–439
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in complexity from simple juxtaposition of the two cell membranes
(Fig. 7) to a structured region of highly convoluted interdigitations
with canaliculi containing a finely granular material (Fig. 8). Ions must
cross this narrow extracellular space driven by the electrochemical
gradients established by the transport systems specific to each cell
type.
4. Discussion
Our electrophoretic and sucrose gradient centrifugation analyses
of M. amazonicum gill microsomes reveal a noticeable alteration in
protein expression pattern between shrimps held in fresh water and
those acclimated to 21‰ salinity. The redistribution of Na,K-ATPase
molecules in membrane fractions of different densities may reflect
vesicle trafficking between intracellular compartments and the
plasma membrane, and is also found in the gills of salinity-acclimated
M. olfersi (Mendonça et al., 2007). Recently, Jayasundara et al. (2007)
identified a putative binding site for a regulatory protein from the ‘14-
3-3’ family in a Na,K-ATPase α-subunit from the posterior gills of the
shore crab Pachygrapsus marmoratus. Members of this family bind to
target proteins regulating their translocation between cytoplasmic or
endoplasmic reticulum localities and the plasma membrane (Dough-
erty and Morrison, 2004; Mackintosh, 2004).
The kinetic parameters estimated for K-phosphatase activity in gill
microsomes from M. amazonicum whether salinity acclimated or
maintained in fresh water were similar except for a 1.8-fold lower
ouabain affinity in the latter (Table 1). In contrast, the kinetic
parameters for the same enzyme from the related species M. olfersi
acclimated to 21‰ differ significantly from those held in freshwater,
which suggests the expression of different isoenzymes during salinity
adaptation in this species (Mendonça et al., 2007). Two α-subunit
isoforms have been identified in some crustacean species (Lucu and
Towle, 2003; Lovett et al., 2006b; Jayasundara et al., 2007) and,
apparently, different isoenzymes are also expressed in crab gill tissue
(Harris and Bayliss, 1988; Genovese et al., 2004; Masui et al., 2005).
Although Na,K-ATPase subunits are expressed in multiple forms in
vertebrate tissues, the kinetic differences among isoenzymes are often
subtle; recent evidence suggests that one of their major functional
distinctions resides in their specific interactions with other proteins
(reviewed by Pressley et al., 2005). Thus, the eventual expression of a
different Na,K-ATPase isoenzyme in M. amazonicum gill tissue mightnot be related to marked kinetic alterations, but rather to the
activation of distinct regulatory mechanisms.
Consistent with ATPase activity data (Santos et al., 2007), the K-
phosphatase specific activity of the Na,K-ATPase in gill microsomes
from M. amazonicum in fresh water was about 2-fold less than that
estimated forM. olfersi (Furriel et al., 2001). Despite such differences, a
similar decrease of about 2–2.5 fold in K-phosphatase specific activity
has been demonstrated in response to salinity acclimation in both
species (Mendonça et al., 2007). InM. rosenbergii, however, there is no
significant variation in gill Na,K-ATPase activity after long-term
exposure to seawater (Wilder et al., 2000).
The decreased K-phosphatase specific activity in M. amazonicum
gill tissue is accompanied by a drastic reduction in α-subunit
expression, as demonstrated by quantitative image analysis of the
immunoreactive bands detected by Western blot. Long-term salinity
acclimation of M. amazonicum also leads to a marked reduction in gill
Na,K-ATPase α-subunit mRNA expression (Faleiros and McNamara,
unpublished data). Salinity-induced variations in α-subunit expres-
sion (Lucu and Flik, 1999; Masui et al., 2005; Lovett et al. 2002, 2006b)
and mRNA levels (Luquet et al., 2005; Chung and Lin, 2006; Li et al.,
2006; Serrano et al., 2007; Jayasundara et al., 2007) also occur in crab
gill tissue. However, α-subunit expression varies little in M. olfersi
(Mendonça et al., 2007), Callinectes sapidus (Towle et al., 2001) and
Scylla paramamosain (Chung and Lin, 2006), despite considerable
alterations in gill Na,K-ATPase specific activity.
The K-phosphatase activity of the M. amazonicum gill enzyme was
stimulated by K+ with an apparent affinity several times greater than
that for NH4+, each ion stimulating activity up to nearly maximal values
(Table 1), independently of acclimation salinity, as seen for M. olfersi
(Furriel et al., 2001; Mendonça et al., 2007) and C. danae (Masui et al.,
2003, 2005). Although the ATPase activity of some crustacean gill Na,
K-ATPases is synergistically stimulated by K+ and NH4+ (Masui et al.,
2002; Gonçalves et al., 2006; Garçon et al., 2007), including those from
M. olfersi (Furriel et al., 2004) andM. amazonicum (Santos et al., 2007),
a similar effect has not been noted for K-phosphatase activity (Masui
et al., 2003; Furriel et al., 2004; Mendonça et al., 2007), in agreement
with the present findings.
The expression and/or activity of phosphate-hydrolyzing enzymes
other than the Na,K-ATPase was stimulated in M. amazonicum gill
tissue in response to salinity acclimation as also seen in M. olfersi
(Mendonça et al., 2007). In contrast to the complete inhibition of
pNPPase activity by ouabain in gill microsomes from shrimps held in
fresh water, the ouabain-insensitive, ethacrynic acid-sensitive
pNPPase activity found in salinity-acclimated M. amazonicum gill
microsomes suggests the presence of Na- and/or K-ATPases. Interest-
ingly, gill microsomes fromM. amazonicum held in freshwater exhibit
low activities of these ATPases when ATP is the substrate (Santos et al.,
2007), suggesting a preference for the natural substrate that may have
impaired detection with pNPP. Na- and K-ATPase activities have been
demonstrated in the gill tissue of M. amazonicum from northern
Venezuela (Proverbio et al., 1990), and in other crustaceans (Wanson
et al., 1984; Gilles and Péqueux, 1985; Harris and Bayliss, 1988; Masui
et al., 2005). However, while the expression of such enzymes may be
related to the long-term response to osmotic challenge (Masui et al.,
2005; Mendonça et al., 2007), their exact location in the gill
epithelium remains unknown and their contribution to the osmor-
egulatory response is unclear. V-ATPase activity is virtually absent in
salinity-acclimatedM. amazonicum gill microsomes when using pNPP
as a substrate. However, low V-ATPase activity is seen in fresh-water
held shrimpwhen using ATP (Santos et al., 2007), reinforcing previous
suggestions that the V-ATPase does not hydrolyze pNPP (Furriel et al.,
2001; Mendonça et al., 2007).
Fig. 8. Gill epithelium ultrastructure in the freshwater shrimp, M. amazonicum.
Transverse section through a complex interface between a pillar cell base (b) and an
intralamellar septal cell (s), revealing a region of intricate interdigitation (⁎) with
narrow canaliculi (arrows). Na+ is proposed to move down its electrochemical gradient
between the two cell types across this junctional complex, driven by the Na,K-ATPase
present in the septal cell invaginations (i). Scale bar=1 µm.
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Adult M. amazonicum from the population studied here are strong
hyper-regulators in fresh water, maintaining hemolymph osmolality
and [Na+] around 400 mOsm/kg H2O and 120 mmol Na+ L−1,
respectively (Augusto et al., 2007). They also survive well in saline
media, hyperosmoregulating up to 20‰, the isosmotic point corre-
sponding to 663 mOsm/kg H2O (=22.1‰) (Augusto et al., 2007).
Hemolymph [Na+], hyper-regulated up to the iso-ionic point at
309 mmol L−1 (=14‰), and conforming at higher salinities, is less
well regulated (Augusto et al., 2007). Immediately on transfer from
fresh water to 21‰ salinity, external Na+ (294 mmol L−1) is about 2.5
times greater than hemolymph [Na+], favoring passive Na+ influx in
addition to active uptake via the gill Na,K-ATPase. The decrease in gill
Na,K-ATPase activity in M. amazonicum after long-term exposure to
21‰, reducing active Na+ capture and energy expenditure, is
consistent with the species' osmoregulatory pattern.
The gill epithelial architecture of M. amazonicum from fresh water
disclosed here corroborates the current palaemonid gill model
(McNamara and Torres, 1999). As a working hypothesis, we propose
that, during uptake from fresh water in this intimately coupled, pillar
cell-septal cell system, after penetrating the fine cuticle into the
subcuticular space, Na+ would move across the amplified surface of
the apical pillar cell membrane into the apical flange cytosol via Na+/
NH4+ and/or Na+/H+ antiporters. V-ATPase activity has been identified
in M. amazonicum gill homogenates (Santos et al., 2007), and the
potential difference resulting from H+ extrusion may also drive Na+
influx through apical Na+ channels. These various transport mechan-
isms would sustain the uphill transfer of Na+ into the apical pillar cell
cytoplasm. Na+ would then diffuse away down its concentration
gradient to the basal pillar cell cytosol where it crosses the narrow
extracellular space into the septal cell cytoplasm via Na+/NH4+
antiporters or Na+ channels on either side of the fine interdigitating
junctions between the pillar cell bases and the intralamellar septal
cells. Again moving down its concentration gradient, Na+ would then
reach the mitochondria-rich cytoplasmic projections created by the
septal cell invaginations, and be actively transported up its gradient by
the Na,K-ATPase into the lumen of the narrow extracellular invagina-
tions (McNamara and Torres, 1999). Bulk fluid flow would then
transfer the Na+-rich fluid into the hemolymph down the Na+
concentration gradient. K+ may recycle locally across the invagination
membranes.
The crab gill Na,K-ATPase can transport NH4+ in exchange for Na+
(Towle and Holleland, 1987), and NH4+ can substitute for K+ in
stimulating the M. amazonicum gill enzyme, which is also synergis-
tically stimulated by K+ and NH4+ (Santos et al., 2007). This would
provide a ready source of NH4+ for uphill Na+ exchange across the apical
pillar cell membrane. Whether NH4+ originates from the protonation
and transport of hemolymph NH3 by the septal cells or whether the
pillar cells themselves produce considerable NH3/NH4+ is uncertain.
The H+ exchanged apically for Na+ may derive from local hydration of
CO2 by carbonic anhydrase. Significant Na+ flux into the hemolymph
directly across the lower pillar cell flange seemsunlikely, since this
membrane is not amplified, and the driving force would be uphill and
diffusive alone. Hemolymph NH3 may diffuse across this membrane,
however. A paracellular pathway for Na+ influx also seems unlikely
given the desmosome-like macula and the lengthy septate junction
between adjacent flange membranes.
During salinity acclimation, this complex transport system is
strongly down-regulated, particularly Na,K-ATPase activity. Whether
the putative transporters at the apical and other interfaces become
internalized in cytoplasmic vesicles is not clear. However, in the
similar diadromous species, M. olfersi, substantial ultrastructural
modifications take place such as a reduction in the number and
length of the apical pillar cell evaginations (McNamara and Lima,
1997), reduced septal cell mitochondrial volume, spatial isolation of
septal cell mitochondria by membrane stacking (Freire and McNa-
mara, 1995) and decreased invagination membrane/mitochondrial
proximity (McNamara and Torres, 1999). Such changes would reduce
Na+ movement across all epithelial cell membranes and limit the
driving force of the Na,K-ATPase by restricting substrate availability.
The present findings in M. amazonicum aid in comprehending the
molecular processes involved in long-termmodulation of Na,K-ATPase
activity in palaemonid shrimp gills in response to salinity acclimation.
Apparently, the decrease in enzyme specific activity derives from
various regulatory mechanisms that act in concert, including reduced
protein expression, trafficking of enzyme molecules and ultrastruc-
tural rearrangements. Some of these mechanisms may be species-
specific, since in M. olfersi, a species exhibiting comparable osmotic
and ionic regulatory capability, the similar decrease in gill Na,K-
ATPase derives from the expression of a different isoenzyme and/or
post-translational modifications rather than from a substantial
decrease in α-subunit expression. Better comprehension of these
processes in crustacean gills will benefit from future investigations of
gene expression.
Acknowledgements
This investigation was supported by research grants from the
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), the
Conselho de Desenvolvimento Científico e Tecnológico (CNPq), and
the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), and constitutes part of M Sc dissertations submitted by NMB
and ROF to the Graduate Program in Comparative Biology, FFCLRP, USP.
KCS Firmino received an undergraduate scholarship from FAPESP and
a M Sc scholarship from CNPq, NMB and ROF M Sc scholarships from
CAPES, DCM a post-doctoral scholarship from FAPESP, and FAL and
JCM research scholarships from CNPq. All experiments conducted
comply with currently applicable state and federal laws. The alpha-5
antibody was obtained from the Developmental Studies Hybridoma
Bank developed under the auspices of the NICHD and maintained by
the University of Iowa, Department of Biological Sciences, Iowa City, IA
52242. We thank Nilton Rosa Alves and Susie Keiko Teixeira for
technical assistance.
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