J Comp Physiol B (1999) 169: 25±37
Ó Springer-Verlag 1999
ORIGINAL PAPER
D. Weihrauch á W. Becker á U. Postel á S. Luck-Kopp
D. Siebers
Potential of active excretion of ammonia in three different
haline species of crabs
Accepted: 14 October 1998
Abstract Isolated perfused gills of stenohaline crabs
Cancer pagurus adapted to seawater, brackish wateradapted euryhaline shore crabs Carcinus maenas and
freshwater-adapted extremely euryhaline Chinese crabs
Eriocheir sinensis were tested for their capacity to excrete
ammonia. Gills were perfused with haemolymph-like
salines and bathed with salines equal in adaptation
osmolality. Applying 100 lmol á l)1 NH4Cl in the perfusion saline and concentrations of NH4Cl in the bath
that were stepwise increased from 0 to 4000 lmol á l)1
allowed us to measure transbranchial ¯uxes of ammonia
along an outwardly as well as various inwardly directed
gradients. The gills of all three crab species were capable
± to di€erent extents ± of active excretion of ammonia
against an inwardly directed gradient. Of the three crab
species, the gills of Cancer pagurus revealed the highest
capacity for active excretion of ammonia, being able to
excrete it from the haemolymph (100 lmol á l)1 NH‡
4)
through the gill epithelium against ambient concentrations of up to 800 lmol á l)1, i.e. against an eightfold
gradient. Carcinus maenas and E. sinensis were able to
actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of
E. sinensis exhibited the greatest capacity to resist in¯ux
at very high external concentrations of up to
4000 lmol á l)1. We consider the observed capacities for
excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect
themselves by burying themselves in the sediment,
where, in contrast to the water column, concentrations
of ammonia have previously been reported that greatly
D. Weihrauch (&) á D. Siebers á U. Postel á S. Luck-Kopp
Biologische Anstalt Helgoland in der Stiftung Alfred-WegenerInstitut fuÈr Polar- und Meeresforschung, Notkestrasse 31,
D-22607 Hamburg, Germany
W. Becker
Zoologisches Institut und Museum,
UniversitaÈt Hamburg, Martin-Luther-King-Platz 3,
D-20146 Hamburg, Germany
increase haemolymph levels. Electrophysiological results
indicate that the permeabilities of the gill epithelia are a
clue to understanding the species-speci®c di€erences in
active excretion of ammonia. During the invasion of
brackish water and freshwater, the permeabilities of the
body surfaces greatly decreased. The gills of marine
Cancer pagurus exibited the greatest permeability (ca.
250 mS cm)2), thus representing practically no in¯ux
barrier for ions including NH‡
4 . We therefore assume
that C. pagurus had to develop the strongest mechanism
of active excretion of ammonia to counteract in¯ux. On
the other hand, freshwater-adapted E. sinensis exhibited
the lowest ion permeability (ca. 4 mS cm)2) which may
reduce passive NH‡
4 in¯uxes at high ambient levels.
Key words Active excretion of ammonia á Crab gill á
Eriocheir sinensis á Cancer pagurus á Carcinus maenas
Abbreviations FW fresh weight á Gte transepithelial
conductance á DPNH3 partial pressure gradients
PNH3 partial pressure of non-ionic ammonia á PDte
transepithelial potential di€erences á S salinity
TAmm total ammonia á TRIS tris±(hydroxymethyl)±
aminomethane
Introduction
Like most aquatic animals, crustaceans are, with few
exceptions, ammoniotelic. Their nitrogenous metabolic
end-products are mainly excreted in the form of ammonia (NH3 ‡ NH‡
4 ). The major proportion of ammonia excretion occurs through the gills, whereas antennal
and maxillary glands play a minor role (Binns and Peterson 1969; Cameron and Batterton 1978; Kormanik
and Cameron 1981; Claybrook 1983; Regnault 1987).
The mechanism of ammonia release across crustacean
gills is considered to be, in the main, a simple di€usion
of non-ionic NH3 along its gradient (Kormanik and
Cameron 1981). Experimental evidence for at least
partial excretion of ammonia in its ionic form, NH‡
4 , has
26
been published; evidence supporting the assumption of
facilitated outward di€usion of the hydrophilic NH‡
4
molecule across the branchial epithelium has been observed in ®sh and arthropods (Armstrong et al. 1981;
PeÂqueux and Gilles 1981; Pressley et al. 1981; Evans and
Cameron 1986; Lucu et al. 1989; Siebers et al. 1995).
Recent studies on isolated perfused gills of the shore
crab Carcinus maenas conducted by Weihrauch et al.
(1998) showed that under physiologically meaningful
concentration gradients of total ammonia (TAmm),
branchial excretion of TAmm proceeded mostly as NH‡
4.
Furthermore, ammonia was actively excreted against a
gradient of TAmm and the gradient of the partial pressure
of NH3 (PNH3). With regard to a certain inconsistency of
the ®ndings, e.g. active release of TAmm utilizes iontransporting proteins, proceeding at increased rates in
anterior gills (Weihrauch et al. 1998) with signi®cantly
reduced capabilities of active ion absorption (PeÂqueux
and Gilles 1978, 1981; PeÂqueux and Chapelle 1982; Siebers et al. 1986; Winkler 1986), experiments were designed to clarify this question. In addition, the present
work is devoted to the question as to whether active
excretion of TAmm is a capacity restricted to the
branchial epithelium of the shore crab only, or whether
this also occurs in other crabs. Therefore, active excretion of TAmm was studied in anterior and posterior gills
of three di€erently haline crabs, the stenohaline crab
Cancer pagurus, the moderately euryhaline crab Carcinus maenas, and the extremely euryhaline Chinese crab
Eriocheir sinensis. While previous experiments (Weihrauch et al. 1998) were performed with symmetrical salines (248 mmol á l)1 NaCl) on the internal and external
gill surface of the shore crab, the present experiments
employ salinity regimes resembling natural conditions.
maenas inhabits shallow coastal water areas. It tolerates salinities
down to approximately 8&, including tidal ¯uctuations in estuaries. The shore crab is less capable of osmoregulation than the
Chinese crab.
Before experimental use, the crabs were adapted for at least 1
month to the maintenance conditions in the laboratory. Determinations of TAmm in haemolymph and urine and excretion of ammonia in intact crabs were performed on crabs adapted to the
respective salinities mentioned above. In order to avoid moulting,
light periods were reduced to 8 h per day. Temperature was kept at
16 °C. Crabs were fed 3 times a week with small pieces of bovine
heart.
Analysis of haemolymph ammonia
Haemolymph samples (ca. 1 ml) were obtained by puncturing the
arthrodial membrane at the base of a walking leg and collected in
refrigerated Eppendorf tubes using a sterilized syringe and a
0.90 ´ 40 mm needle. After centrifugation (5 min at 5000 g, 4 °C)
supernatant samples of clear serum were split into 2 aliquots of
0.4 ml which were then diluted with haemolymph-like salines to a
®nal volume of 2 ml, sealed and deep-frozen at )70 °C for no
longer than 1 week prior to measurements. For determination of
ammonia, an ammonia-sensitive electrode was used throughout the
experiments. All haemolymph samples were taken 24 h after
feeding.
Ammonia excretion of intact crabs
For measuring total net e‚ux of TAmm intact crabs were placed
separately into beakers ®lled with 1 l of freshwater (E. sinensis),
brackish water of 10& (Carcinus maenas) or seawater of 35&
(Cancer pagurus). External media (16 °C) were continuously aerated during the experiment. After stabilization of ammonia e‚ux
within 1 h (data not shown; see also Hunter and Kirschner 1986),
the external media used so far were replaced by the same media
followed by a sampling period of 1 h. Triplicates of 2 ml for the
ammonia determination were taken before (control) and after the
experiment. Samples were analysed for their concentration of TAmm
on the day of experimentation or were sealed and immediately
frozen at )70 °C for no longer than 1 week prior to determination
of ammonia.
Materials and methods
Crabs
Adult crabs Cancer pagurus of approximately 8±15 cm carapace
width were caught in the North Sea near the island of Heligoland.
Adult males were kept in seawater of 35& salinity (S) in 600-l
basins (ca. 1 crab per 40 l). The water was continuously aerated,
®ltered and recirculated using the seawater circulation system of the
institute. Shore crabs Carcinus maenas were obtained from a ®sherman in Kiel Bay, Baltic Sea. In the laboratory, adult males of
approximately 5±7.5 cm carapace width were maintained in 200-l
aquaria and adapted to brackish water of 10& S (ca. 1 crab per 20
l). The water was continuously aerated and ®ltered over gravel.
Chinese crabs E. sinensis were caught by a ®sherman in the river
Eider (approximately 0.5& S) and transferred to the institute. In
the laboratory, the adult males with a carapace width of 4±7 cm
were kept in running freshwater in 500-l tanks (ca. 1 crab per 20 l).
Of the three crab species, Cancer pagurus is restricted to oceanic
waters. It is the most stenohaline species and possesses only limited
capacities for regulation of the osmolalities of the body ¯uids
(Harris and Bayliss 1988). The Chinese crab E. sinensis inhabits
freshwater rivers and lakes. Only for reproduction do adults migrate down the rivers into the tidal estuaries to spawn in brackish
water or seawater. Due to its high capacity of active osmoregulatory ion absorption across the gills, the Chinese crab is extremely
euryhaline (Gilles 1975; Gilles and PeÂqueux 1986; Gilles et al. 1988;
Onken and Graszynski 1989). The euryhaline shore crab Carcinus
Preparation and perfusion of gills; determination of ammonia
Crabs were killed by destroying the ventral ganglion using a spike
which was pressed through the ventral side of the body wall. The
carapace was lifted and the gills were removed. The gills were
perfused according to the method described by Siebers et al. (1985)
with a ¯ow rate of 0.135 ml min)1. During perfusion, transepithelial potential di€erences (PDte) were monitored using a millivolt
meter (type 197, Keithley, Cleveland, USA) connected with the
perfusate and the bath solutions by means of two Ag/AgCl electrodes (type 373-S7, Ingold, Frankfurt/Main, Germany). PDte was
measured in individual gills to control the success of the preparation. Constant PDte was established within an incubation period of
30 min. The external bath and the perfusion solution were then
replaced, followed by a sampling period of 30 or 60 min. Duplicate
samples of 2 ml were taken from the bath (original volume 30 ml)
and from the perfusate after passage through the gill. In order to
continue the experiment with the same gill the procedure was repeated with modi®ed salines (changes in the NH4Cl concentrations
or addition of ionic transport inhibitors).
In¯ux experiments were carried out by increasing step by step
the external concentrations of NH4Cl. Between the steps, no preincubation periods of 30 min were included. The samples were
analysed for their content of TAmm on the day of experimentation
or were sealed and immediately frozen at )70 °C prior to measurements of ammonia concentrations within the following week.
At the end of the experiment, the gill was cut above the clamp,
27
dried under light pressure between two sheets of soft paper
(Kleenex) and weighed [Fresh weight (FW)]. Determination of
TAmm in the samples was achieved using a gas-sensitive NH3
electrode (the electrode sensitivity and the procedures of calculation of concentrations have been reported in detail by Weihrauch
et al. 1998).
tration of ammonia in the sample at the end of the experiment
(lmol á l)1); V is the volume of the external bath or perfusate (ml); t
is the sampling period (h) and FW is the fresh weight of the gill (g).
In order to calculate PNH3 it was necessary to determine the
relation of the concentrations of NH3 and NH‡
4 , which in solution
are present in a pH-dependent equilibrium, by use of the Henderson-Hasselbalch-equation for ammonia:
Determination of gill resistance and calculation
of transepithelial conductance
pH ˆ pK ‡ log
After removing the gills, single gill lamellae were isolated and split
according to the method described by Schwarz and Graszynski
(1989). In this way, a single epithelial layer covered by an apical
cuticle was obtained. The preparation was mounted in an Ussing
chamber modi®ed after De Wolf and Van Driessche (1986), allowing determinations of area-speci®c (0.02 cm2 or 0.01 cm2)
short-circuit currents (Isc) and transepithelial resistances (Rte). The
chamber compartments were continuously superfused with salines
at a rate of 0.5 ml min)1 by means of a peristaltic pump. For
measurements of the PDte, Ag/AgCl electrodes were connected via
agar bridges (3% agar in 3 mol á l)1 KCl) with the chamber compartments (distance of the preparation less than 0.1 cm). A second
pair of Ag/AgCl electrodes, connected through agar bridges, served
as current electrodes to short-circuit the PDte with an automatic
clamping device (VCC 600, Physiologic Instruments, San Diego,
USA). The transepithelial conductance (Gte) was calculated as
Gte ˆ 1/Rte. For further details of electrophysiological measurements, see Riestenpatt et al. (1996).
Salines and chemicals
The ionic composition of the salines used to perfuse isolated gills
and to determine transepithelial resistances in gill half lamellae
mounted in an Ussing chamber resembled in vivo conditions. The
salines for the apical bath (isolated perfused gills) and the apical
superfusion media (isolated gill half lamella mounted in an Ussing
chamber) contained (mmol á l)1): 431.5 NaCl, 5 CaCl2, 5 KCl,
4 MgCl2, 2 NaHCO3, 2.5 tris±(hydroxymethyl)±aminomethane
(TRIS) for experiments on gills of Cancer pagurus, 150 NaCl,
5 CaCl2, 5 KCl, 4 MgCl2, 2 NaHCO3, 2.5 TRIS for experiments
on gills of Carcinus maenas, and 7 NaCl, 2 CaCl2, 0.3 KCl,
2 MgCl2, 1 NaHCO3, 2.5 TRIS for experiments on gills of E.
sinensis.
Corresponding to haemolymph composition, the perfusion salines (isolated perfused gills) and the basolateral superfusion salines
(isolated gill half lamella mounted in an Ussing chamber) contained
(mmol á l)1): 431.5 NaCl, 5 CaCl2, 5 KCl, 4 MgCl2, 2 NaHCO3,
2.5 TRIS for experiments on gills of Cancer pagurus, 300 NaCl,
5 CaCl2, 5 KCl, 4 MgCl2, 2 NaHCO3, 2.5 TRIS for experiments
on gills of Carcinus maenas, and 248 NaCl, 5 CaCl2, 5 KCl,
4 MgCl2, 2 NaHCO3, 2.5 TRIS for experiments on gills of E.
sinensis. Immediately before use, 2 mmol á l)1 glucose was added to
the basolateral salines in all experiments, and the pH of all salines
was adjusted to 7.8 (HCl).
Amiloride hydrochloride and ouabain were purchased from
Sigma; CsCl was purchased from Merck (Darmstadt, Germany).
The ammonia standard (0.1 mol á l)1) was obtained from Orion
Research Incorporated (Boston, USA), and all other chemicals
were of analytical grade and purchased from Merck.
Calculations
Transbranchial ¯uxes of ammonia (JAmm) were expressed in
lmol á l)1 g FW)1 h)1 and calculated according to:
…Cbeg ÿ Cend † V
…1†
1000 t FW
where Cbeg is the concentration of ammonia in the sample
(lmol á l)1) at the beginning of the experiment; Cend is the concenJAmm ˆ
‰NH3 Š
‰NH‡
4Š
…2†
According to a nomogram published by Cameron and Heisler
(1983), pK amounts to 9.53 at 20 °C and a concentration of
450 mmol á l)1 NaCl. PNH3 were calculated according to Eq. (3):
PNH3 ˆ ‰NH3 Š=a
…3†
where a is the solubility coecient and [NH3] the concentration of
NH3.
For
our
calculations
we
used
the
value
a ˆ 43.67 mmol á l)1 torr)1 for plasma at 20 °C (Cameron and
Heisler 1983). NH3 partial pressure gradients (DPNH3) were calculated from the di€erences in PNH3 between external and internal
media. All results are presented as means ‹SEM. Di€erences between groups were tested using the paired Student's t-test. Statistical signi®cance was assumed for P < 0.05.
Results
Concentrations of TAmm in haemolymph
and rates of excretion by intact crabs
In order to apply physiological haemolymph concentrations of TAmm in experiments with isolated perfused gills
of the three crab species, it is necessary to analyse these
concentrations in the haemolymph of intact crabs
adapted to the ambient salinities described in Materials
and methods. Haemolymph concentrations of TAmm
amounted to 80 ‹ 13 lmol á l)1 (n ˆ 7) in Cancer pagurus (adapted to 35& S), to 99 ‹ 28 lmol á l)1 (n ˆ 6)
in Carcinus maenas (adapted to 10& S), and to
116 ‹ 18 lmol á l)1 (n ˆ 10) in freshwater E. sinensis.
Haemolymph concentration in seawater-acclimated
Carcinus maenas was slightly reduced (83 ‹
12 lmol á l)1; n ˆ 10) compared to that in brackish wateradapted crabs. Excretion of TAmm by intact crabs during
an observation period of 1 h was variable. The highest
rates ± 336 ‹ 46 nmol g FW)1 h)1 (n ˆ 7) ± were observed in Cancer pagurus (adapted to 35& S). In the other
two crab species excretion rates were similar but lower:
136 ‹ 30 nmol g FW)1 h)1 (n ˆ 7) in Carcinus maenas
(adapted to 10& S), and 123 ‹ 18 nmol g FW)1 h)1
(n ˆ 11) in fresh water E. sinensis.
Production and release of ammonia
by anterior and posterior gills
Fluxes of TAmm resulting from the production and release of ammonia from the gill metabolism were determined over a period of 1 h in isolated gills perfused and
bathed in salines which did not contain ammonia at the
beginning of the experiment. Posterior gills are the three
posterior-most pairs of gills of the three crab species;
anterior gills are the three gill pairs located directly be-
28
fore the posterior gills. The highest rates of total release
of ammonia were observed in Cancer pagurus
(12.3 ‹ 3.5 lmol g FW)1 h)1, n ˆ 6, in anterior gills
and 7.8 ‹ 1.9 lmol g FW)1 h)1, n ˆ 6, in posterior
gills) (Fig. 1). Less than half these rates were found in
Carcinus maenas and E. sinensis, with the lowest rates in
the gills of the latter crab. The release of ammonia was
nearly the same in anterior and posterior gills of
E. sinensis. In the gills of the shore crab, release in the
anterior gills was slightly increased compared with that
in the posterior gills, and in Cancer pagurus release in
anterior gills greatly exceeded the rates observed in
posterior gills. In all three crabs branchial liberation of
ammonia was higher and more signi®cant on the apical
side. Between 65% and 80% of the total ammonia
originating from branchial metabolism of the crabs was
excreted across the apical side of the gill epithelium
which faces the ambient bathing saline.
Potential of active excretion of ammonium ions
across anterior and posterior gills
Cancer pagurus
In order to ®nd out the potential of active excretion of
ammonium ions across anterior and posterior gills of the
three crab species, isolated gills were perfused with
100 lmol á l)1 NH4Cl. This concentration is an approximate mean of TAmm determined in the haemolymph of
intact crabs. Concentrations of total ammonia in the
bathing saline of the gills were increased step by step
from 0 lmol á l)1 at the beginning of the experiment to a
®nal concentration of 4000 lmol á l)1. At the beginning
of each 0.5 h experimental period the perfusion and the
Fig. 1 Production and release rates of ammonia by anterior and
posterior gills of seawater-adapted Cancer pagurus (n ˆ 6), brackish
water-adapted Carcinus maenas (n ˆ 6), and freshwater-adapted
Eriocheir sinensis (n ˆ 4). Gills were bathed and perfused with salines
which contained no ammonia at the beginning of the experiment.
Ionic compositions of the bathing and perfusion solutions resembled
in vivo conditions (see Materials and methods). Data represent
means ‹ SEM
bathing medium were exchanged. This experimental setup allowed us to measure the ¯uxes of TAmm along
varying outwardly and inwardly directed gradients in
each individual gill.
In order to determine the transbranchial ¯uxes of
TAmm, we considered the changes of ammonia in the
perfusion media between the beginning of each experimental period (100 lmol á l)1 NH4Cl) and its end 0.5 h
later. The reason for this procedure is that the experiments comprised ammonia regimes between perfusion
and bathing medium that reached very high bath concentrations of up to 4000 lmol á l)1. Changes of TAmm as
a result of transbranchial ¯ux would alter such a high
concentration only slightly and thus render an exact determination dicult. In addition, the possibility cannot
be excluded that the preferred release of ammonia originating from gill metabolism (Fig. 1) to the apical side
would comprise a potential source of error if the calculation were based on changes in the bathing medium.
When the apical concentration of TAmm was
0 lmol á l)1 NH4Cl, an e‚ux of ammonia along the
100 lmol á l)1 gradient directed from the perfusate to the
bath was observed in Cancer pagurus which amounted to
15.2 lmol g FW)1 h)1 in the anterior gills and to
13.5 lmol g FW)1 h)1 in the posterior gills (Fig. 2A,
and in detail Fig. 2B). The e‚ux rates decreased only
slightly with increasing starting concentrations in the
bath. At bath concentrations of 100 lmol á l)1 NH4Cl,
the gills were exposed to symmetrical concentrations of
TAmm. Under these conditions a net e‚ux was observed
amounting to 12.0 lmol g FW)1 h)1 in the anterior gills
and to 9.7 lmol g FW)1 h)1 in the posterior gills
(Fig. 2A, and in detail Fig. 2B).
It was of interest to ®nd out whether or not the gills
of Cancer pagurus were capable of outward transport of
TAmm against an initial concentration gradient. As
shown in Fig. 2A and B, both gill pairs were capable of
net outward transfer of TAmm up to an external concentration of 400 lmol á l)1 NH4Cl, representing an external/internal gradient of 300 lmol á l)1. Even at an
external concentration of 800 lmol á l)1 NH4Cl corresponding to a gradient of 700 lmol á l)1, the anterior gills
of
still
showed
a
net
e‚ux
of
TAmm
2.6 lmol g FW)1 h)1, whereas in posterior gills at this
29
Fig. 2A±F Fluxes of total ammonia (TAmm) across anterior and
posterior gills of seawater-adapted Cancer pagurus, brackish wateradapted Carcinus maenas, and freshwater-adapted E. sinensis. Gills
were perfused with salines containing 100 lmol á l)1 NH4Cl. Concentrations of NH4Cl in the bathing salines increased stepwise from 0 to
4000 lmol á l)1. A, C, and E shows the ¯uxes determined over the
complete concentration regime. B, D, and F show ¯uxes in detail
observed under bathing media concentrations between 0 and
800 lmol á l)1 NH4Cl. Data represent means ‹ SEM. A, B n ˆ 7
(anterior) and n ˆ 9 (posterior gills). C, D n ˆ 7 (anterior) and
n ˆ 8 (posterior gills). E, F n ˆ 7 (anterior) and n ˆ 6 (posterior
gills)
30
unfavourable concentration gradient the capacity of
ammonia excretion was exhausted. A net in¯ux was
observed which amounted to 4.9 lmol g FW)1 h)1.
Transbranchial net in¯uxes increased along with internal/external
concentration
gradients
up
to
4000 lmol á l)1 NH4Cl; however, this occurred at reduced concentration-gradient-dependent rates in anterior compared to posterior gills. It is obvious that
especially the anterior gills are capable of inhibiting in¯ux of ammonia even when the unfavourable gradient
reaches very high and unphysiological levels.
Carcinus maenas
Comparable net e‚uxes of ammonia along an internal
to external gradient of 100 lmol á l)1 NH4Cl were observed in the shore crab C. maenas (Fig. 2C, D). Under
symmetrical conditions (100 lmol á l)1 NH4Cl on both
sides of the epithelium) net e‚uxes amounted to
9.5 lmol g FW)1 h)1
in
anterior
gills
and
6.7 lmol g FW)1 h)1 in posterior gills showing a higher
capacity of ammonia excretion in anterior gills of this
crab as well as in those of Cancer pagurus. Net e‚uxes
against initial external to internal concentration gradients were reduced in comparison with the capacity of
C. pagurus; the concentration gradient at which the capacity of the gills was exhausted was smaller. Given an
external concentration of 400 lmol á l)1 NH4Cl (a concentration gradient of 300 lmol á l)1), net e‚uxes in
anterior gills amounted to 1.5 lmol g FW)1 h)1,
whereas at this concentration gradient a small net in¯ux
of 0.2 lmol g FW)1 h)1 was observed in posterior gills.
At the extremely high external concentration of
4000 lmol á l)1 (a gradient of 3900 lmol á l)1), both anterior and posterior gills of the shore crab showed net
in¯uxes comparable to the rates observed in the posterior gills of C. pagurus.
l á l)1 (a gradient of 700 lmol á l)1). Under this concentration gradient the net e‚ux across the posterior gills
was 1.0 lmol g FW)1 h)1. When the concentration
gradients were increased stepwise up to 3900 lmol á l)1
net in¯uxes of TAmm remained comparably small. In¯uxes at the highest concentration gradient were
17.8 lmol g FW)1 h)1
in
anterior
gills
and
11.0 lmol g FW)1 h)1 in posterior gills.
Under symmetrical conditions (100 lmol á l)1 NH‡
4 in
the bath and in the perfusate) decreases in the concentrations of TAmm in the internal perfusate were calculated for 30 mg standard gills. In Cancer pagurus
perfusate concentrations of TAmm decreased from
100 lmol á l)1 to 64.3 ‹ 5.9 lmol á l)1 (n ˆ 8) in anterior gills and to 71.0 ‹ 4.5 lmol á l)1 (n ˆ 9) in posterior gills. Since the perfusate passes the gill only once
these results show that concentrations of TAmm were
reduced by 35.7% during a single passage of perfusate
through anterior gills and 29.0% through posterior gills.
Similar results were obtained in Carcinus maenas. In
addition, the anterior gills of this crab exhibited an increased capacity of active excretion of NH‡
4 compared
to the posterior gills; the anterior gills reduced internal
levels of TAmm by 34.3% (n ˆ 7) and the posterior gills
by 20.7% (n ˆ 8). In contrast to these two species, the
posterior gills of E. sinensis showed a higher capacity of
reducing perfusate levels of TAmm when compared to the
anterior gills. In the Chinese crab, perfusate concentrations of TAmm were reduced by only 3.9% (n ˆ 7) in
anterior gills and by 14.8% (n ˆ 6) in posterior gills.
Permeability of the branchial epithelia
Ion permeabilities of the branchial epithelia of the three
species were analysed in order to ®nd out potential
Eriocheir sinensis
Remarkable di€erences in the ammonia excretory capability and pattern were noted in the Chinese crab
E. sinensis compared to those in Cancer pagurus and
Carcinus maenas. When no ammonia was present in the
ambient bath (a gradient of 100 lmol á l)1 NH4Cl
directed from perfusate to bath), the e‚uxes of TAmm
were 3.1 lmol g FW)1 h)1 in anterior gills and
8.8 lmol g FW)1 h)1 in posterior gills (Figs. 2E, F).
With 100 lmol á l)1 NH4Cl applied in a symmetrical
mode, net e‚uxes were 1.2 lmol g FW)1 h)1 in anterior
gills and 4.6 lmol g FW)1 h)1 in posterior gills.
The capacities of the anterior gills to resist in¯ux of
ammonia were already reached at a bath concentration
of 200 lmol á l)1 NH4Cl. At this concentration gradient
(100 lmol á l)1) a net in¯ux of 0.2 lmol g FW)1 h)1 was
observed. Posterior gills were capable of net ammonia
excretion up to an ambient concentration of 800 lmo-
Fig. 3 Transepithelial conductance in isolated half lamellae of gills of
seawater-adapted Cancer pagurus, brackish water-adapted Carcinus
maenas, and freshwater-adapted E. sinensis. Half lamellae of anterior
and posterior gills were mounted in a modi®ed Ussing chamber. Data
represents means ‹ SEM with the number of experiments given in
parentheses
31
correlations with transepithelial ¯uxes of TAmm described in the following paragraphs. Permeabilities in
terms of the transepithelial conductances, Gte, were investigated in isolated split lamellae of single platelets of
anterior and posterior gills. Split lamellae were mounted
in a modi®ed Ussing chamber and superfused on the
apical and basolateral surface with salines resembling
in vivo salinity regimes. The highest permeabilities were
found in Cancer pagurus, 282 ‹ 50 mS cm)2 (n ˆ 13)
in anterior gills and 253 ‹ 61 mS cm)2 (n ˆ 13) in
posterior gills (Fig. 3). Nearly ®ve times reduced values
of Gte were observed in Carcinus maenas, 62 ‹
7 mS cm)2 (n ˆ 12) in anterior gills and 41 ‹
9 mS cm)2 (n ˆ 9) in posterior gills. The smallest Gte
was observed in the Chinese crab E. sinensis, 4 ‹
1 mS cm)2 (n ˆ 6) in anterior gills and 4 ‹ 1 mS cm)2
(n ˆ 19) in posterior gills.
Branchial excretion of total ammonia
against a pre®xed gradient in C. pagurus
Isolated anterior gills of Cancer pagurus were perfused
and bathed with a saline (431.5 mmol á l)1 NaCl) which
resembles the ionic composition of seawater and haemolymph. At the beginning of the experiment concentrations of TAmm were 100 lmol á l)1 in the perfusate and
150 lmol á l)1 in the external bath. This regime represents an initially inwardly directed gradient of TAmm and
DPNH3. After an experimental period of 0.5 h the perfusate concentration of TAmm had decreased from
100 lmol á l)1 to 54.2 ‹ 16.1 lmol á l)1 (expressed for a
standard gill of 30 mg FW) and the bath concentration
had increased from 150 lmol á l)1 to 171.8 ‹
5.0 lmol á l)1 (n ˆ 6) (Fig. 4A).
During the experiment pH changed from 7.8 to
7.83 ‹ 0.01 (n ˆ 6) in the perfusate. In the bath pH
remained constant at 7.8. The PNH3 calculated by use of
these
®gures
decreased
from
41.9 ltorr
to
19.5 ‹ 2.6 ltorr in the perfusate and increased from
62.8 ltorr to 75.3 ‹ 1.9 ltorr (n ˆ 6) in the bathing
saline (Fig. 4B). The initial DPNH3 was inwardly directed
and increased from 20.9 ltorr to 55.8 ‹ 4.2 ltorr
(n ˆ 6) while still being directed inwardly.
In this experiment ¯ux rates of TAmm were determined
using the observed changes in the perfusate and in the
bath. Ammonia was excreted against the gradients of
concentration of TAmm and PNH3. The excretion rate
calculated from the decrease of TAmm of the perfusate
was 16.3 ‹ 5.6 lmol g FW)1 h)1, and the excretion rate
calculated from the increase of TAmm of the bath was
43.6 ‹ 10.1 lmol g FW)1 h)1 (n ˆ 6). The di€erences
in the ¯ux rates of 27.3 ‹ 4.9 lmol g FW)1 h)1 are
considered to represent the amounts of ammonia originating from gill metabolism (see Fig. 1).
E€ects of ion transport inhibitors on active excretion
of NH‡
4 across anterior gills of Cancer pagurus
Fig. 4 Bath and perfusate changes of TAmm (A) and NH3 partial
pressures (B) during 0.5 h of branchial excretion of TAmm against a
pre-®xed gradient in anterior gills of Cancer pagurus. At the beginning
of the experiment, concentrations of TAmm were 100 lmol á l)1 in the
perfusate and 150 lmol á l)1 in the external bath. Data represent
means ‹SEM calculated from n ˆ 6 experiments
In order to obtain information on the nature of the
process of active excretion of ammonium ions shown to
be present in the anterior gills of C. pagurus (for posterior
gills see Fig. 2), known inhibitors of active osmoregulatory ion uptake were applied. At the beginning of the
perfusion, experiment concentrations of TAmm were
100 lmol á l)1 in the perfusate and 150 lmol á l)1 in the
external
bath.
Basolaterally
applied
ouabain
(5 mmol á l)1), a potent and speci®c inhibitor of Na+/
K+ATPase (Skou 1965), not only reduced excretion of
TAmm completely but even resulted in a net in¯ux. This
e€ect was partially reversible following omission of the
inhibitor (Fig. 5A). Basolaterally applied Cs+
(10 mmol á l)1), a speci®c inhibitor of K+ channels (Van
Driessche and Zeiske 1980; Riestenpatt et al. 1996), reduced the active excretion of NH‡
4 only slightly and insigni®cantly by approximately 15% without reversibility
upon omission of the antagonist (Fig. 5B). Apically
32
Fig. 5A±C E€ects of ion transport inhibitors on active excretion of
NH‡
4 across anterior gills of Cancer pagurus. At the beginning of the
experiment, concentrations of TAmm were 100 lmol á l)1 in the
perfusate and 150 lmol á l)1 in the external bath. A Basolateral
application of 5 mmol á l)1 ouabain (n ˆ 5, P < 0.001); B basolateral application of 10 mmol á l)1 Cs+ (n ˆ 3, di€erences are not
signi®cant), and C apical application of 0.1 mmol á l)1 amiloride
(n ˆ 5, P < 0.05). Data represent means ‹ SEM
added amiloride (0.1 mmol á l)1), an inhibitor of Na+/
H+ exchangers (Ahearn 1996; Towle 1997; Towle et al.
1997), signi®cantly inhibited the active excretion of NH‡
4
by approximately 30% in a reversible mode (Fig. 5C).
Discussion
Concentrations of haemolymph ammonia
and excretion of TAmm by intact crabs
It is the aim of this investigation to describe the capability of active excretion of total ammonia recently detected in the shore crab Carcinus maenas under
comparative aspects in three brachyuran species. The
crabs chosen for this work are quite di€erent in their
degree of euryhalinity and osmoregulatory abilities (see
Materials and methods). In experiments on isolated
perfused anterior and posterior gills and gill half
lamellae, the haemolymph-like internal salines and the
salines employed in the bathing media of the gills
resembled in vivo conditions with respect to ionic composition and osmolality. Potentially detected di€erences
in excretion of TAmm within the three species have to be
considered in relation to haemolymph ammonia concentrations, since these may represent a component of
the force driving ammonia out of the haemolymph
through the gill and into the external bath. Remarkable
di€erences in haemolymph TAmm between the species
considered were not found. Concentrations in the three
species amounted to approximately 100 lmol á l)1, a
comparatively low ®gure.
In C. maenas acclimated to 35&, we found slightly
and insigni®cantly reduced concentrations of haemolymph TAmm compared to specimens adapted to 10& S.
This result con®rms the above-mentioned assumption of
similar haemolymph TAmm in all three crab species and
implies that haemolymph TAmm in individuals adapted
to di€erent salinities over prolonged periods of several
months is not salinity dependent. This situation is different in euryhaline ®shes and crustaceans during sudden hypo-osmotic stress, in which, according to Gilles
(1979), amino acids leak out of the cells followed by an
increase in the levels of blood ammonia.
Our experiments on C. maenas have shown that
concentrations of haemolymph TAmm are highly sensitive to the feeding regime. Haemolymph concentrations
of TAmm were 333 ‹ 10 lmol á l)1 (n ˆ 3) 4 h after
feeding with strong decreases in the following hours and
days (Weihrauch, personal observation). Therefore all
experiments were performed exactly 24 h after feeding.
Similar concentrations of haemolymph TAmm were detected in few other crustacean species, 100 lmol á l)1 in
the freshwater cray®sh Cherax destructor (Fellows and
Hird 1979), and 97 lmol á l)1 in Carcinus maenas from
Maine or the Baltic Sea which were adapted to 15&
seawater (Lucu et al. 1989). Higher concentrations of
haemolymph TAmm of up to 1 mmol á l)1 were published
for other aquatic crustaceans (for review see Greenaway
1991).
In terrestrial crustaceans facing restricted conditions
of water availability, even higher concentrations of
haemolymph TAmm have been reported: 1.7 mmol á l)1 in
Cardisoma guanhumi (Horne 1968), 1.2 mmol á l)1 in
non-excreting and 2.1 mmol á l)1 in excreting Geograpsus
grayi (Varley and Greenaway 1994), 2±3 mmol á l)1 in
the same species (Greenaway and Nakamura 1991), 2±
4 mmol á l)1 in Gecarcoidea natalis (Greenaway and
Nakamura 1991), 1.6 mmol á l)1 in Cadisoma carnifex
(Wood et al. 1986), 1.5 mmol á l)1 in Porcellio scaber
(Wieser and Schweizer 1972).
The excretion rates of TAmm in intact crabs (between
123 nmol g FW)1 h)1 and 336 nmol g FW)1 h)1) were
comparable to those observed in other crustaceans species, i.e. 142 nmol g FW)1 h)1 in Cancer antennarius
adapted to seawater (Hunter and Kirschner 1986);
131 nmol g FW)1 h)1 in Carcinus maenas (Binns 1969);
183 nmol g FW)1 h)1 in freshwater-adapted E. sinensis
(Florkin et al. 1964), and 149 nmol g FW)1 h)1 in seawater-acclimated blue crabs Callinectes sapidus (Cameron 1986). It is remarkable that Cancer pagurus
33
exhibited more than a twofold increased excretion rate
of TAmm compared with the other two crab species, a
result indicative of a high metabolic utilization of protein.
Production and release of ammonia by gills
Our experiments on the release of metabolic TAmm by
isolated gills in absence of ammonia in the bathing and
perfusion media showed without exception a clear
preference of liberating metabolic TAmm to the environment in anterior and posterior gills of all three crab
species studied despite the di€erent experimental salinity
regimes applied (Fig. 1). Excretion rates of TAmm were
increased approximately twofold in Cancer pagurus
compared with the rates measured for the gills of
Carcinus maenas and E. sinensis. This result obtained
from the gills con®rms the assumption of high metabolic
utilization of protein in Cancer pagurus derived
from excretion rates of TAmm in intact specimens (see
preceding paragraph). In line with this assumption
are the ®ndings by King et al. (1985) who compared the
activities of phosphate-dependent glutaminase in the
gills of two species of Cancer (C. irrogatus and C. borealis) as well as in Carcinus maenas. Speci®c activities of
phosphate-dependent glutaminase were between
)1
120 lmol NH‡
min)1
and
140 lmol
4 mg protein
)1
)1
‡
NH4 mg protein min in the two Cancer species, but
)1
only about 30 lmol NH‡
min)1 in Car4 mg protein
cinus maenas. The ®ndings are suggestive of a particularly high dependence of branchial metabolism on
amino acids in Cancer, and correspond to the increased
excretion rates of TAmm observed in gills (Fig. 1) and
intact specimens of C. pagurus.
Ecological relevance of active excretion of TAmm
The ®nding that all three crab species are capable of
active excretion of TAmm poses the question as to
whether this physiological gill function is of ecological
signi®cance. Traditionally, ammonia excretion in
aquatic animals is considered to be a passive process
driven by di€usion along the gradient, since environmental concentrations are kept low as a result of bacterial nitri®cation of ammonia to nitrite and nitrate
followed by absorption by autotrophs. We consider
this view as justi®ed in pelagic animals colonizing the
water column of aquatic biota where low concentrations of TAmm occur. According to Korole€ (1983), the
)1
amounts of NH‡
in oxygen4 rarely exceed 5 lmol á l
ated, unpolluted seawater. In contrast, benthic and
interstitial animals are often faced with higher ambient
concentrations of TAmm present especially in anoxic,
deep stagnant water and pore water during periods of
high mineralization following collapses of phytoplankton blooms. Such phenomena are observed in the
North Sea especially in late summer when in calm pe-
riods vertical density strati®cation is low, accompanied
by low oxygen supply which inhibits nitri®cation processes.
In recent years several projects have been conducted
by marine institutes to investigate nutrient concentrations in water samples of the southern North Sea with
respect to the potential e€ects of eutrophication. At
several stations very high concentrations of TAmm ± up
to 20 lmol á l)1 ± were determined in the water column
(Brockmann et al. University of Hamburg, personal
communication). At stations of the Wadden Sea around
the North Sea island Sylt, pore water concentrations of
)1
NH‡
at 4 cm below
4 were approximately 100 lmol á l
the sediment surface in November 1993 and approximately 140 lmol á l)1 at 12±14 cm below the sediment
surface in August 1993 (L.-A. Meyer-Reil, Hiddensee,
Germany, personal communication). This area is colonized by various crustaceans, among them Carcinus
maenas and Cancer pagurus (Herrmann et al. 1998). In
general, nutrient concentrations in coastal areas are increased by the nutrient loads brought in by the rivers.
Concentrations of 200±400 lmol á l)1 NH‡
4 and in few
cases ca. 800 lmol á l)1 have been determined by U.
Brockmann (University of Hamburg, personal communication) in pore waters of the lower river Elbe and the
Wadden Sea of the Elbe estuary. In sediment samples
taken in September 1981 at two stations in the Gullmar
Fjord, considered by the authors as likely to be representative of the coastal waters of southwest Scandinavia,
Enoksson and Samuelsson (1987) determined NH‡
4
concentrations between ca. 120 lmol á l)1 and
)1
170 lmol á l at a sediment depth of 4 cm and concentrations between ca. 110 lmol á l)1 and 300 lmol á l)1 at
9 cm sediment depth. Lohse et al. (1993) reported that
ammonium in interstitial waters of the southeastern
North Sea was always the prevailing inorganic nitrogen
component, showing a trend of lower concentrations at
o€shore stations to higher values along the German and
Danish coast. At the Esbjerg (25 m water depth) and
Heligoland Rinne (39 m water depth), stations NH‡
4
concentrations up to 170 lmol á l)1 were detected in
August 1991 about 7 cm below sediment surface. Extremely high values of 2800 lmol á l)1 were found at the
Heligoland Bight station (19 m water depth) 9 cm below
sediment surface and these seemed to increase further
with depth.
The three species of benthic crabs investigated in this
work hide under stones with small water exchange or
bury themselves in the sediment, where at low rates of
ambient water exchange they permanently excrete ammonia. Considering haemolymph concentrations of
TAmm of ca. 100 lmol á l)1 in these species, it is obvious
that under speci®c environmental circumstances the
crabs may actually face ambient concentrations of TAmm
that exceed haemolymph levels. Exposed to inwardly
directed gradients of ammonia an adaptive protection
against in¯ux, i.e. a mechanism for excretion of metabolic ammonia against its gradient, must have evolved in
these species.
34
The potential of active transbranchial excretion
of ammonium ions in relation to the permeability
of the epithelium
Crustacean gills are the main organs for exchanges between the body and its environment. They play a central
role in osmo- and ionoregulatory active ion uptake
(Kirschner 1979; Towle 1981; Lucu 1990), exchanges of
respiratory gases (Burnett and McMahon 1985; BoÈttcher
et al. 1991; BoÈttcher and Siebers 1993), excretion of nitrogenous metabolic end-products (Kormanik and
Cameron 1981), and regulation of the acid-base status of
the haemolymph (Truchot 1979; Perry and Laurent 1990;
Henry and Wheatly 1992). The permeability characteristics of these body appendages and the speci®cally designed
salt transport mechanisms greatly determine their capacity to maintain haemolymph homeostasis during adaptation to brackish and limnic environments.
Figure 3 shows the permeability of the branchial
epithelium in terms of Gte, measured in individual splitgill half lamellae mounted in a modi®ed Ussing chamber. The data indicate that in Cancer pagurus the permeability of the gills is very high, and the epithelium
thus very leaky. The permeability of the gills of Carcinus
maenas is about ®ve times lower than that in Cancer
pagurus; however, the gills are still leaky. Compared to
Cancer pagurus, the permeability of the gills of E. sinensis
is nearly 60 times lower, and the epithelium has to be
considered as moderately tight. This classi®cation for
Carcinus maenas and E. sinensis corresponds with ®ndings published by Onken and Siebers (1992), and Riestenpatt et al. (1996), and also with the epithelial
characteristics proposed by Erlij and Martinez-Palermo
(1978) and Schultz (1979).
The permeability of the three crab species corresponds to their ecological requirements. C. pagurus is
stenohaline and restricted to marine habitats, where its
haemolymph is isosmotic with the ambient medium.
Since passive salt losses are considered to be low, the
permeability of the body surface including the gills may
be small, and the necessity of active branchial ion uptake
may also be low. Due to Carcinus maenas inhabiting
brackish water and having to counterbalance passive
salt losses, the permeabilities of the body surface were
reduced along with the development of the capability of
active salt absorption across the gills. Of the three species under consideration, fresh-water Chinese crabs are
capable of maintaining the largest salinity gradients
between the body ¯uids and the ambient water by
greatly reducing the permeability of the body surface
and developing a high capacity for active branchial ion
uptake (Gilles 1975).
In this context we argue that at localities with increased levels of TAmm Cancer pagurus must prevent
di€usive in¯ux of NH3 and NH‡
4 across its very leaky
gill epithelium that represents practically no in¯ux barrier for NH‡
4 . Therefore this crab has developed a highly
e€ective mechanism of active excretion of TAmm to
counteract TAmm in¯ux (Fig. 2A, B).
The Chinese crab is also able to excrete NH‡
4 actively, but less e€ectively than Cancer pagurus (Fig. 2E,
F). In contrast to C. pagurus, the moderately tight
branchial epithelium of freshwater-adapted E. sinensis is
much less permeable for di€usive in¯uxes of NH‡
4 . The
Chinese crab may thus face problems excreting metabolic TAmm across this barrier. Since di€usive in¯uxes of
NH‡
4 in this crab are restricted by the tightness of the
gills, the mechanism of excretion of TAmm must only
counterbalance di€usive in¯uxes of NH3 and secure the
e‚ux of metabolic TAmm. The epithelium of Carcinus
maenas is about ten times more leaky than that of E.
sinensis and about ®ve times less leaky than that of
Cancer pagurus. This leakiness of the branchial epithelium of the shore crab facilitates ± as in C. pagurus ± the
in¯ux of ambient ammonia. The highly e€ective N-excretion observed in Carcinus maenas may be an adaptation to its epithelial permeability property. The high
ion permeability of the gills of Cancer pagurus and
Carcinus maenas may thus explain the ®ndings that the
former was able to actively excrete TAmm against an
eightfold internally directed gradient and that the latter
was capable of active excretion against a fourfold gradient (Fig. 2B, D).
The degree of epithelial leakiness may also provide
the key for understanding the species-speci®c di€erent
in¯ux rates of TAmm at very high external concentrations
of up to 4000 mol á l)1. High in¯ux rates at this ambient
concentration facilitated by high permeability were observed in Cancer pagurus and Carcinus maenas, while in
E. sinensis in¯uxes were roughly one half the rates found
in the other two species (Fig. 2A, C, E).
Without additional experiments using inhibitors of
the transporting structures that play a role in ion and/
or NH‡
4 translocation, it is not possible to discriminate
between active NH‡
4 excretion, facilitated di€usion of
NH‡
4 and simple di€usion of NH3 within the in¯uxes
of TAmm at very high ambient concentrations. Because
of the large DPNH3 we do anticipate that a signi®cant
portion of the in¯uxes under these conditions consists
of NH3 di€usion.
Active excretion of Tamm against a pre®xed gradient
across the anterior gills of Cancer pagurus:
e€ects of ion transport antagonists
In order to investigate the mechanism for active excretion of TAmm, three potential antagonists of ion-transporting membrane proteins were tested in isolated
perfused anterior gills of C. pagurus; ouabain, Cs+ and
amiloride. The anterior gills were chosen because of
their high capacities of active excretion of TAmm. Due to
their similar charges and hydrated ionic radii (1.45 AÊ) it
+
is expected that NH‡
4 can cross membranes via K translocating pathways such as the ouabain-sensitive
Na+/K+ATPase, Cs+-sensitive K+ channels and the
perNa+/K+/2Cl) co-transporter. However, NH‡
4
meabilities of K+-selective channels are considerably
35
lower than corresponding K+ permeabilities (see
Knepper et al. 1989). On the other hand, several ion
channels with little selectivity among monovalent cations have been described which may be as comparably
+
selective for NH‡
(Latorre and Miller 1983).
4 as for K
‡
Interactions of NH4 with the amiloride-sensitive Na+/
H+ exchanger have been shown by Kinsella and
Aronson (1981) in rabbit renal microvillus membrane
vesicles.
Bathing anterior gills of C. pagurus with
150 lmol á l)1 NH‡
and perfusing them with
4
100 lmol á l)1 resulted in an actively mediated decrease
of TAmm in the perfusion medium by 46% during a
single passage of perfusate through the gill along with
increases in the bath (Fig. 4A). The increase of DPNH3
across the epithelium during the experiment (Fig. 4B)
increased the force driving the in¯ux of NH3. The active
excretion of NH‡
4 , however, also proceeded against this
adverse movement.
The completely reversible reduction of the active excretion of TAmm across anterior gills by ouabain
(Fig. 5A) shows that the Na+/K+ATPase plays a
dominant role in this excretory mechanism. Previously
we showed that in posterior gills of Carcinus maenas
symmetrically exposed to 100 lmol á l)1 NH‡
4 , ouabain
inhibited the active e‚ux of TAmm only by 60%, a result
implying the presence of a second active component in
this process (Weihrauch et al. 1998). We therefore conclude that in Cancer pagurus the Na+/K+ATPase is the
only energy consuming component of the active excretion of TAmm. This conclusion is in line with the result
that under the comparatively small inwardly directed
)1
gradient of TAmm of 50 lmol NH‡
inhibition of
4 ál
+
+
Na /K ATPase by ouabain resulted in a net in¯ux
(Fig. 5A). Correspondingly, studies in isolated perfused
mammalian renal tubules have emphasized the possible
importance of the Na+/K+ATPase in renal ammonium
transport (Garvin et al. 1985); this enzyme can function
as a primary NH‡
4 pump, oriented to mediate active
NH‡
uptake
into
cells (reviewed by Knepper et al.
4
1989).
The employment of basolateral Cs+ also showed a
di€erence in the mechanism of active excretion of TAmm
between Carcinus maenas and Cancer pagurus. As reported by Weihrauch et al. (1998), active transbranchial
excretion of NH‡
4 was inhibited by 60% by basolateral
Cs+ in the shore crab, showing the involvement of
basolateral K+ channels in this process. As obvious
from Fig. 5B, Cs+ applied in the basolateral saline of
perfused gills of Cancer pagurus inhibited the active excretion of NH‡
4 only slightly, insigni®cantly and without
reversibility. Obviously, basolateral Cs+-sensitive K+
channels are not involved in this process in C. pagurus.
The reversible apical amiloride inhibition of active e‚ux
of NH‡
4 by 29% (Fig. 5C) shows that besides amiloridesensitive Na+/H+ (NH‡
4 ) exchangers and/or cation
pores, other structures must play a role in releases of
NH‡
4 across the apical membranes of the branchial epithelium, including the cuticle. Corresponding to our
®ndings on amiloride e€ects are the results reported by
Hunter and Kirschner (1986) who found nearly identical
inhibitory e€ects of amiloride on the excretion of TAmm
by intact Cancer antennarius.
In conclusion, the remarkably similar haemolymph
concentrations of TAmm in the three crab species investigated (ca. 100 lmol á l)1) are determined by the rate of
metabolic production of ammonia and the rate of
branchial excretion. Since excretion is driven by the internal/external concentration gradients of TAmm and
partial pressure of PNH3, environmental concentrations
of TAmm exceeding haemolymph levels may theoretically
result in in¯uxes of toxic ammonia. This situation may
indeed occur in the habitat of benthic organisms which
protect themselves from in¯uxes by developing mechanisms of excreting TAmm against the gradient, i.e. actively and thus energy consuming. When brackish and
limnic aquatic environments were settled by formerly
marine species, the animals developed reduced permeabilities of their body surfaces and mechanisms of
active uptake of ions to compensate for gradient-driven
ion losses. In each of the di€erently haline crab species,
Cancer, Carcinus and Eriocheir, the capacity of active
excretion of ammonia was developed in relation to the
permeability of the species-speci®c epithelium to prevent
in¯uxes of ammonia at elevated environmental levels.
This deduction is re¯ected in the very leaky epithelium
of Cancer in which active excretion of TAmm is much
higher than in Eriocheir with its moderately tight epithelium, wheras active ammonia excretion in the leaky
epithelium of Carcinus is moderate.
Acknowledgements This article is based on a doctoral study performed by Dirk Weihrauch at the Faculty of Biology, University of
Hamburg. The ®nancial support of this work by the Deutsche
Forschungs Gemeinschaft (Si 295/2-3) is gratefully acknowledged.
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