Ammonia excretion in aquatic and terrestrial crabs
1 Department of Biology, Division of Animal Physiology, University of
Osnabrück, D-49076 Osnabrück, Germany
2 Morlab, School of Biological Sciences, University of Bristol, BS8 1UG,
UK
3 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
* Author for correspondence at present address: Department of Biology, Division of Animal Physiology, Universität Osnabrück, D-49076 Osnabrück, Germany (e-mail: Weihrauchblues{at}gmx.net)
Accepted 24 September 2004
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Summary |
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Key words: ammonia excretion, ammonia transporter, crab, exocytosis, Rhesus-like protein, Na+/K+-ATPase
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The ammonia problem |
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In crustaceans, for example in the lobster Homarus americanus
(Young-Lai et al., 1991) and
the crayfish Pacifastacus leniusculus
(Harris et al., 2001
),
elevated ammonia levels in low-salinity media disrupt ionoregulatory function.
Exposure of the green shore crab Carcinus maenas to 1 mmol
l1 total ammonia leads to increased ion permeability and
salt flux across the gill; higher concentrations reduce both variables
(Spaargaren, 1990
). In fish,
branchial gas exchange and oxidative metabolism are disturbed by excess
ammonia (Wilkie, 1997
).
An effective ammonia detoxification or excretion system is, therefore,
essential to maintain cellular functions, and to keep cellular and body fluid
ammonia levels within a tolerable range. In most species, including mammals
(Cooper and Plum, 1987), fish
(Wood et al., 2002
) and
aquatic crabs (Cameron and Batterton,
1978
; Weihrauch et al.,
1999
), the ammonia concentration of the body fluids is typically
low (50400 µmol l1;
Table 1). Concentrations
exceeding 1 mmol l1 total ammonia
(NH3+NH4+) are usually toxic to mammalian
cells (Hrnjez et al., 1999
).
In crustaceans, environmental exposure of ammonia is lethal at relatively low
doses. For instance, LC50 after 96 h of exposure was determined in
the crayfish Orconectes nais at 186 µmol l1
NH3 (Hazel et al.,
1982
), in the Sao Paulo shrimp Penaeus paulensis at 19
µmol l1 NH3 and 0.307 mmol
l1 total ammonia
(Ostrensky et al., 1992
) and
in the redtail prawn Penaeus penicillatus 58 µmol
l1 NH3 and 1.39 mmol l1 total
ammonia (Chen and Lin,
1992
).
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Mammals accrue ammonia both from metabolism and as an influx to the hepatocytes from the gastrointestinal tact. This ammonia is detoxified in the urea cycle, an energy-consuming process, by incorporation into the less-toxic urea. Crustaceans are largely ammonotelic, aquatic species exclusively so, and in water excrete their nitrogenous waste directly to the environment as highly soluble ammonia.
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Origin of ammonia in crustaceans |
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Organs of ammonia excretion in aquatic crabs
In aquatic crabs, primary urine is formed via ultrafiltration in
the antennal gland, which is thought to play the key role in regulation of
body water and divalent cations (e.g. Mg2+, Ca2+;
Mantel and Farmer, 1983), but
not to contribute significantly to the excretion of nitrogenous waste products
(Regnault, 1987
). For
instance, in the blue crab Callinectes sapidus, <2% of total
ammonia is excreted in the urine via the antennal gland system
(Cameron and Batterton,
1978
).
The main site for ammonia excretion by aquatic crabs is the
phyllobranchiate gill (Claybrook,
1983; Kormanik and Cameron,
1981
; Regnault,
1987
), featuring a single-cell-layered epithelium covered by an
ion-selective cuticle (Avenet and Lignon,
1985
; Lignon,
1987
; Onken and Riestenpatt,
2002
, Weihrauch et al.,
2002
). The gills of aquatic crabs are multifunctional organs. In
addition to their function in excretion of nitrogenous waste products, they
are also responsible for respiratory gas exchange
(Burnett and McMahon, 1985
),
regulation of acidbase balance
(Henry and Wheatly, 1992
) and
osmoregulatory ion transport (Towle,
1981
; Lucu, 1990
,
Riestenpatt et al., 1996
,
Towle and Weihrauch, 2001
).
Several transporters and enzymes putatively linked and involved in ammonia
transport have been shown to be present in the branchial epithelium of crabs,
as summarized in Fig. 2 and
Table 2.
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Ammonia excretion in aquatic crabs
In solution, both forms of ammonia, non-ionic ammonia (NH3) and
the ammonium ions (NH4+) exist in a pH-dependent
equilibrium. As a weak base (pK 9.48 at 20°C and NaCl=250 mmol
l1; Cameron and Heisler,
1983
) and at a physiological pH of pH 7.8, 98% of total ammonia
exists in the ionic form NH4+, whereas only 2% is
present as non-ionic NH3. However, the higher lipid solubility of
NH3 makes it more diffusible through phospholipid bilayers.
Kormanik and Cameron (1981
)
reported that ammonia excretion of seawater adapted blue crabs Callinectes
sapidus occurred mainly by diffusion of non-ionic NH3. An
excretion mechanism based predominately on NH3 diffusion is not
likely, however, because membrane permeability of NH3 is much lower
than that of CO2 (Knepper et
al., 1989
). Indeed, some plasma membranes of animal epithelia are
relatively impermeable to NH3 as shown for frog oocytes
(Burckhardt and Frömter,
1992
), the renal proximal straight tubules
(Garvin et al., 1987
) and
colonic crypt cells (Singh et al.,
1995
). Accordingly, other authors have obtained experimental
evidence for at least partial excretion of ammonia in its ionic form
(NH4+) in Callinectes sapidus
(Pressley et al., 1981
) and
Carcinus maenas (Lucu,
1989
; Siebers et al.,
1995
).
Studies on isolated perfused gills of several aquatic crabs showed that
ammonia can be excreted actively against a 48-fold inwardly directed
ammonia gradient across both the anterior and the posterior gills to a similar
degree despite their different morphological and physiological characteristics
(Copeland and Fitzjarrell,
1968; Goodmann and Cavey,
1990
; Weihrauch et al.,
1998
,
1999
;
Towle and Weihrauch, 2001
)
(Fig. 3). Under physiologically
relevant conditions, the potential for active branchial ammonia excretion is
significantly greater in the marine Cancer pagurus than in
freshwater-acclimated Chinese mitten crabs Eriocheir sinensis,
despite the much larger ionic conductance of Cancer pagurus gills
(
250280 mS cm2) compared with that of
Eriocheir sinensis gills (
4 mS cm2)
(Fig. 4). It is noteworthy that
the posterior gills of Carcinus maenas (thought to play the dominant
role in osmoregulatory NaCl uptake) and also the anterior gills (thought to be
primarily responsible for gas exchange) are equally capable of active ammonia
excretion (Weihrauch et al.,
1999
).
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Ecological relevance of active ammonia excretion in aquatic crabs |
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By contrast, benthic and interstitial animals are often faced with higher
ambient ammonia concentrations. High ammonia is especially prevalent in
anoxic, deep stagnant water and pore water during periods of high
mineralization following collapse of phytoplankton blooms. For example,
several investigations of pore water composition in the North Sea showed
considerable concentrations of ammonia in a range between 100 and 300 µmol
l1, but also up to 2500 µmol l1 in
49 cm sediment depth (Enoksson and
Samuelsson, 1987; Lohse et
al., 1993
).
Like most aquatic crab species, Carcinus maenas, Cancer pagurus and Eriocheir sinenis are benthic-living animals, hiding under stones or burying themselves in the sediment for long periods, for example, during low tide or in the winter season. Under conditions where crabs are situated at sites with low rates of ambient water exchange, plus the fact that the animals produce and excrete metabolic ammonia, the concentration of the ambient ammonia can reach high values.
Considering hemolymph ammonia concentrations of 100 µmol
l1 (Weihrauch et al.,
1999
; Table 1) of
which less than 5 µmol l1 exist in the gaseous form
NH3, these crabs may encounter ambient NH3 and/or
NH4+ concentrations exceeding those in their hemolymph.
While NH3 diffuses along its partial pressure gradient across the
exposed epithelia, NH4+ follows its electrochemical
gradient by either paracellular diffusion or NH4+
permeable channels and transporters (see
Table 2). An adaptive
protection against net ammonia influxes (i.e. an active mechanism for
excretion of metabolic ammonia against an inwardly directed gradient,
tolerances for high hemolymph ammonia concentrations or efficient
detoxification mechanisms) must, therefore, have evolved.
Branchial ammonia excretion mechanisms in aquatic crabs
In the blue crab Callinectes sapidus, ammonia excretion rates are
correlated with Na+ absorption
(Pressley et al., 1981). The
same result was obtained both for the Chinese crab Eriocheir sinensis
(Péqueux and Gilles,
1981
) and for the shore crab Carcinus maenas
(Lucu et al., 1989
). Studies
employing membrane vesicles from gill epithelia
(Towle and Hølleland,
1987
) and isolated, perfused gills
(Lucu et al., 1989
) indicated
that NH4+ substitutes for K+ in activation of
the ouabain-sensitive Na+/K+-ATPase. In gill sections
from Callinectes sapidus, this Na+/K+-ATPase
was demonstrated to be located in the basolateral membranes of the branchial
epithelial cells (Towle and Kays,
1986
; Towle et al.,
2001
). Complete or partial cDNA sequences for the
-subunit
of Na+/K+-ATPase from crab gills have been published in
GenBank (see Table 2) thus
confirming both its presence in branchial epithelia and its similarity to
-subunits of other species.
Recently, Masui et al.
(2002) showed that the
branchial Na+/K+-ATPase from Callinectes danae
is synergistically stimulated by NH4+ and K+,
increasing its catalytic activity by up to 90%. Masui et al.
(2002
) came to the conclusion
that the two ions bind to different sites of the branchial
Na+/K+-ATPase. This observation was also attributed to
the branchial Na+/K+-ATPase of the freshwater shrimp
Macobrachium olfersii by Furriel et al.
(2004
), who suggested for this
species that at high NH4+ concentrations the pump
exposes a new binding site for NH4+ which, after binding
to NH4+, modulates the activity of the
Na+/K+-ATPase independently of K+ ions.
In the marine crab Cancer pagurus, active branchial excretion of
ammonia is completely inhibited by ouabain, a specific inhibitor of the
Na+/K+-ATPase
(Weihrauch et al., 1999),
suggesting this pump is the only driving force for excretion. However, in the
gills of Carcinus maenas acclimated to brackish water, both
gradient-driven (Lucu et al.,
1989
) and active ammonia excretion
(Weihrauch et al., 1998
) are
only partially inhibited by ouabain, consistent with a second active mechanism
responsible for branchial ammonia extrusion in this species.
The presence of an apically located amiloride-sensitive
Na+/NH4+ exchanger, transporting
NH4+ from the epithelial cell into the ambient medium in
exchange for Na+, has been suggested for Callinectes
sapidus (Pressley et al.,
1981) and for Carcinus maenas
(Lucu et al., 1989
;
Siebers et al., 1995
). Indeed,
branchial mRNA expression of a Na+/H+-antiporter,
putatively transporting also NH4+ ions, was demonstrated
in Carcinus maenas (Towle et al.,
1997
) and in Eriocheir sinensis
(Weihrauch and Towle, 2000
).
However, experiments employing the isolated cuticle from Carcinus
maenas have shown that cuticular Na+ and
NH4+ conductances (Gcut) are inhibited by
apically applied amiloride in a dose-dependent manner, with an inhibitor
constant KamiNa+=0.6 µmol l1 for
sodium ions and KamiNH4+=20.4 µmol
l1 for ammonium ions, respectively
(Onken and Riestenpatt, 2002
;
Weihrauch et al., 2002
).
Differences in KamiNH4+ and
KamiNa+ are not understood yet. One can speculate that
amiloride blocks the passage of cations in the cuticle in a mechanical way
like a plug, rather than by blocking a general cation-binding side. Passage of
smaller ions (like Na+) that carry a larger coat of water molecules
is, therefore, possibly easier to block out by lower amiloride concentrations
than K+ or NH4+ ions, which carry only about
half the number of water molecules around their core. However, according to
these observations, some of the results obtained by applying amiloride to crab
gills (apical) should be interpreted with caution and with special attention
to the concentration of this particular inhibitor.
Further studies on the branchial ammonia excretion mechanism in
Carcinus maenas employing the K+ channel blocker
Cs+ (10 mmol l1) revealed that basolateral (but
not apical) K+ channels play a role in the excretory process
(Weihrauch et al., 1998). In
addition, experiments inhibiting the branchial V-Type H+-ATPase by
adding bafilomycin A1 resulted in a reduction of active ammonia
transport by 66%, identifying the H+-ATPase as the second active
component in the excretory mechanism of the shore crab
(Weihrauch et al., 2002
).
While in Eriocheir sinensis a V-Type H+-ATPase has been
localized to the apical membrane of the gill epithelium
(Onken and Putzenlechner,
1995
), in Carcinus maenas this pump was found
predominantly in the cytoplasm, probably associated with vesicles
(Weihrauch et al., 2001a
).
This latter finding led to the suggestion
(Weihrauch et al., 2002
) of a
vesicular ammonia-trapping mechanism, in which cellular NH3
diffuses into acidified vesicles to be transformed into its
membrane-impermeable ionic form, NH4+. For a directed
excretion, these NH4+-loaded vesicles would then be
transported to the apical membrane for exocytotic release. Such an excretion
mechanism was supported by data showing total inhibition of active ammonia
excretion by blockers of the microtubule network, including colchicine,
thiabendazole and taxol (Weihrauch et
al., 2002
). The resulting hypothetical model of the ammonia
excretion in Carcinus maenas is described in detail in
Fig. 5.
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For crabs (such as the partially limnic Chinese crab Eriocheir
sinensis) that utilize a proton gradient across the apical membrane of
the epithelial cell to accomplish NaCl uptake from highly diluted media, it is
likely that NH3 diffuses across the apical membrane along its
partial pressure gradient, as shown in freshwater rainbow trout
Oncorhynchus mykiss (Wilson et
al., 1994).
Recently, Weihrauch and others (D. Weihrauch, unpublished data) have
sequenced a full-length cDNA coding for a Rhesus-like protein from
Carcinus maenas gills (GenBank Accession number: AF364404), named
RhCM (Rhesus-related protein from Carcinus maenas). In mammals
Rhesus-related proteins, such as RhGK, have been shown to mediate ammonia
(NH3/NH4+), but not K+ or amino
acid transport when functionally expressed in yeast mutants lacking endogenous
ammonia transporters (triple Mep mutant;
Marini et al., 2000
). However,
for this novel Rhesus-like ammonia transporter the detailed transport
characteristics (such as mode of transport or kinetics) have not yet been
defined. A comparison of the deduced secondary structure of the amino acid
sequence of RhCM, and the human ammonia transporter RhGK, showed that 10 out
of 12 predicted transmembrane domains are positioned at identical sites of the
sequence (Fig. 6). The
localization and role of RhCM in branchial ammonia excretion need to be
investigated in detail in further studies. One can speculate that the putative
ammonia transport of crabs is not localized in the apical membrane of the gill
epithelium, because here ammonia (NH3/NH4+)
permeable structures would be of disadvantage allowing ammonia influxes when
the animals are exposed to high external concentrations. The human Rhesus-like
ammonia transporter RhGK (identical to RhCG), has been described to be
localized in the distal tubule and the collecting duct of the kidney in
co-localization with a V-type H+-ATPase
(Eladari et a., 2002
). RhCG
expressed in Xenopus oocytes facilitates a highly specific
NH3 diffusion via a complex electrogenic
NH4+ transport
(Bakouh et al., 2004
). In
addition, Eladari et al.
(2002
) suggested a secondary
active mode of ammonia transport in the distal tubule by acid trapping.
According to this assumption, RhGK would promote the transmembrane passage of
NH3. A similar mechanism would be plausible in the gills of the
shore crab Carcinus maenas, however, RhCM would be co-localized with
the H+-ATPase within the membranes of intracellular vesicles to
support the proposed vesicular acid-trapping mechanisms. Also, a basolateral
localization cannot be excluded, where the putative ammonia transporter might
serve as an overflow valve, transporting ammonia back in to the hemolymph,
when crabs are exposed to high external ammonia concentrations. Under this
condition, intracellular ammonia concentrations might rise to toxic levels due
to a passive influx from the apical side, while the
Na+/K+-ATPase is actively pumping
NH4+ from the hemolymph space into the cytoplasm.
Ammonia directed back into the hemolymph, via RhCM, could probably be
buffered, at least for a short term, by incorporation into proteins, for
instance glutamine or hemocyanins.
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The high degree of conservation with ammonia transporters found in fungi,
bacteria and archaebacteria (20%), as well as the striking homology to
mammalian ammonia transporters (>40%), led to the suggestion that proteins
of the Rh-family play a universal role in ammonia transport
(Fig. 7).
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Ammonia excretion in terrestrial crabs consequences of air exposure
The mechanisms and processes by which air-breathing crustaceans excrete
nitrogenous waste into the terrestrial habitat have been subject to
considerable scrutiny (Greenaway,
1988,
1991
; Wolcott,
1991
,
1992
;
O'Donnell and Wright, 1995
).
Terrestrial crabs appear to tolerate considerably greater hemolymph ammonia
loads than do aquatic species (Table
1). Conversely, Wolcott
(1992
) points out that
diluting ammonia to non-toxic levels in the urine might require an
unsustainable water loss in a land crab.
The probability that branchial NH4+ excretion is
linked to sodium transport, both through apical ion exchangers and the basal
membrane Na+/K+-ATPase, is of special importance to
air-breathing crabs. In all land crabs examined to date, the gills have become
adapted for reabsorption of salt from primary urine directed through the
branchial chamber (Wolcott and Wolcott,
1985,
1991
;
Morris, 2001
), allowing
diffusive NH3 loss and NH4+ extrusion in
exchange for required ions from the urine. Ocypodid crabs seem exceptional in
utilizing the antennal gland for increasing urinary ammonia, although the
gills are required to complete the excretory process
(DeVries and Wolcott, 1993
).
Generally, while the gills are initially bathed with a fluid isosmotic with
the hemolymph, osmotic concentration may decline by as much as 90%
(Wolcott and Wolcott, 1985
;
Varley and Greenaway, 1994
;
Greenaway, 1999
;
Morris et al., 2000
;
Taylor and Greenaway, 2002
;
Morris and Ahern, 2003
). Even
very euryhaline aquatic species do not experience the same range of
osmo-concentration as occurs in the extra-branchial fluid of some land
crabs.
In terrestrial arthropods as a whole, the primary nitrogenous excretory
products are generally purines, whereas in land crabs various mechanisms are
employed to permit the continued excretion of ammonia. The single known
exception is the terrestrial anomuran Birgus latro, which is
purinotelic excreting urate (Greenaway and
Morris, 1989) and guanine
(Greenaway, 2003
). The reasons
for the general persistence of ammonotely may be found by examining a
continuum of extant species in the transition from aquatic to land crab.
Ammonia excretion and air exposure of aquatic crabs
Exposure to air of the aquatic crabs Cancer pagurus and Cancer
productus caused hemolymph ammonia to increase by 25 µmol
h1 (Regnault,
1992) and 26 µmol h1
(deFur and McMahon, 1984
),
respectively. However, in Cancer pagurus this rate of accumulation
was between 15 and 30% of the rate expected by Regnault
(1992
) on the basis of basal
aquatic rates, leading to the suggestion of nitrogen storage in tissues, to
avoid toxic hemolymph ammonia loading. Many crustaceans store nitrogen (N) as
solid urate (for review see Greenaway,
1999
) but this seems to be formed primarily as a result of diet
rather than being of any large significance in
NH3/NH4+ detoxification
(Linton and Greenaway, 1997a
)
except possibly under desiccating conditions in land crabs (below). In
any case, urate formation was ruled out as a significant contribution in
Cancer pagurus (Regnault,
1992
). Ammonia excretion during air exposure of Cancer
pagurus was only 4% of the normal aquatic rate (170190 µmol
kg1 h1) but when re-immersed they
exhibited a very large (50-fold within 5 min) but transient increase to 8860
µmol kg1 h1 (derived from
Regnault 1994
). Thus, nitrogen
storage as NH4+, or as some readily oxidized form, is
apparently a normal response to transient air exposure, as is the subsequent
pulsatile clearance of ammonia on re-immersion. The possibility that urate can
be so rapidly mobilized to NH4+ seems unlikely and
expensive. However, the vesicular sequestration of NH4+
(see above) has hitherto not been considered in this role.
Ammonia excretion in land crabs that immerse
A similar `storageexcretion' is seen in diverse air-breathing crabs
(e.g. Potamonautes warreni,
Morris and van Aardt, 1998;
Austrothelphusa transversa,
Linton and Greenaway, 1995
;
Discoplax hirtipes, Dela-Cruz and
Morris, 1997
; Cardisoma carnifex,
Wood et al., 1986
). [Note:
Cardisoma hirtipes has been revised to Discoplax hirtipes
and Holthuisana to Austrothelphusa
(Davie, 2002
).] Discoplax
hirtipes excretes 99% of its waste as ammonia, but when it is breathing
air the rate of nitrogen loss in the urinary flow is only 0.2 µmol
kg1 h1 and NH3 is volatilized
at a very slow rate (0.4 µmol kg1 h1).
On re-immersion, the ammonia excretion rate is transiently elevated to 1100
µmol kg1 h1 (compared with the normal
300 µmol kg1 h1). Potamonautes
warreni also does not excrete while in air but, on return to water,
excretes ammonia across the gills at 4900 µmol kg1
h1 (compared with the normal rate in water of 70 µmol
kg1 h1). Artificially irrigating the gills
of air-breathing Potamonautes warreni sustained ammonia excretion
(Morris and van Aardt, 1998
).
Gill irrigation appears to be a ubiquitous activity following excursions into
the terrestrial environment (Dela-Cruz and
Morris, 1997
). The requirement to re-immerse, albeit briefly, to
accomplish ammonia excretion via the ancestral branchial mechanisms
may ultimately limit the duration of air-breathing in these amphibious
species. However, Austrothelphusa transversa can spend many months
without access to water (Greenaway and
MacMillan, 1978
) and can forgo ammonia excretion during that time
(Linton and Greenaway, 1995
).
Linton and Greenaway (1995
)
suggested that the near-cessation of nitrogen excretion in A.
transversa implied reduced nitrogen catabolism and temporary nitrogen
storage. The speed and brevity of the excretion pulse in P. warreni
showed that wastes stored during terrestrial forays are rapidly excreted on
return to water. However, it seems unlikely that this store is accumulated as
NH4+/NH3 within the gill epithelium because
hemolymph levels remain low and pH (and therefore
PNH3) remains unchanged
(Adamczewska et al., 1997
)
although the required enzymes may be present
(Linton and Greenaway, 1998
).
Further study is required to determine the storage product but an accessible
intermediate is implicated (such as glutamine rather than, for example,
urate). Apical H+/NH4+ exchange and
V-ATPase-driven cell alkalization have been suggested as likely mechanisms of
transbranchial ammonia transport (Linton
and Greenaway, 1995
), but experimental evidence is required to
confirm this. Again, the involvement of a Rhesus-related ammonia transporter
needs to be evaluated.
Ammonia excretion in terrestrial crabs
Gecarcinid land crabs recycle their urine over the branchial surfaces,
producing a dilute fluid `P' (Wolcott and
Wolcott, 1985; for review
Morris, 2002
). Discoplax
hirtipes, a crab that immerses from time to time, can reduce the NaCl
concentration of the urine by 90%
(Dela-Cruz and Morris, 1997
)
but this ion pumping does not allow ammonia excretion while in air. For
example, while the NH4+ content of `P' of Discoplax
hirtipes is significantly elevated (5 mmol l1) compared
with the hemolymph (Table 1), the rates of urinary and `P' flow slow down to almost zero when the animals
are in air (Dela-Cruz and Morris,
1997
). Thus, ammonia excretion becomes severely limited by urine
flow and consequent `P' production rates.
In Gecarcoidea natalis, a gercarcinid land crab that does not
routinely immerse or have access to pools of water, the primary urine contains
0.36 mmol NH4+ l1, which is less than
in the blood (Table 1).
However, reprocessed `P' contains up to 10.8 mmol l1
(Greenaway and Nakamura,
1991), and this is sufficient to excrete up to 68% of the total
nitrogenous output because `P' production was
450 µl
kg1 h1. This flow rate is much greater
than the 3 µl kg1 h1 in Discoplax
hirtipes, which is unable to sustain ammonia excretion in air
(Dela-Cruz and Morris, 1997
).
The rate of `P' production in Gecarcinus lateralis was >900 µl
kg1 h1, which facilitated an excretion
rate of 20 µmol kg1 h1
(Wolcott, 1991
), compared
with the 25 µmol kg1 h1 rate in
Gecarcoidea natalis (Greenaway
and Nakamura, 1991
). These authors
(Greenaway and Nakamura, 1991
;
Wolcott, 1991
) concluded that
acid trapping in the `P' was not involved and, thus, the outward gradient
across the gill epithelia is not favorable for gaseous NH3
diffusion. In addition, Wolcott
(1991
) measured the urine pH
of Gecarcinus lateralis and Cardisoma guanhumi, and in both
crabs found it to be greater than that of the hemolymph. However, branchial
Na+/H+ exchange would assist in NH3 diffusion
through reciprocal pH changes of intracellular and extra-corporeal fluids. The
gills of Gecarcoidea natalis are highly active in Na+
transport and NH4+ might easily substitute for
K+ in the basal Na+/K+-ATPase
(Morris, 2001
;
Morris and Ahern, 2003
) but
the necessary active transport of NH4+ across the apical
membrane into the `P' remains unresolved. A net exchange of Na+ for
NH4+ (e.g. Pressley
et al., 1981
) would facilitate salt reclamation and nitrogen
excretion, but would be hampered as the external Na+ declined. The
possibility of exocytotic mechanisms and/or involvement of a Rhesus-related
ammonia transporter needs to be evaluated in the excretory processes of these
species.
While this branchial system allows routine excretion of ammonia to air, it
also seems to make NH4+ excretion dependent on the
urinary flow rate, as well as on the extent of ion re-absorption. For example,
in Gecarcoidea natalis, urine and `P' flow can decline to zero under
dry season conditions (Morris and Ahern,
2003) and so this mode of NH4+ clearance
becomes inoperable. In Cardisoma guanhumi the fluid retained within
the abdominal flap (
13.5 mmol l1) is contiguous with
that in the branchial chamber (
6.5 mmol l1) and further
NH4+ excretion may occur via unknown mechanisms
(Wolcott, 1991
) but, even so,
this would be unavailable to land crabs in the dry season. Purine is stored in
large amounts in connective tissue cells throughout the bodies of some land
crabs (Linton and Greenaway,
1997b
). In Gecarcoidea natalis this stored purine is
normally synthesized de novo, from excess dietary nitrogen
(Linton and Greenaway, 1997a
).
Recent data, including enzyme activities and nitrogen utilization (Linton and
Greenaway, 1998
,
2000
) have lead to the
suggestion of a storageexcretion function for the urate accumulated by
G. natalis (Greenaway,
2003
). However, this seems likely to be infrequently called upon
(Linton and Greenaway, 1997a
)
and evidence is required to show that waste amino N is incorporated as well as
dietary N.
Other lineages of terrestrial crabs have not been investigated to the same
extent as the gecarcinids, but at least one air-breathing ocypodid,
Ocypode quadrata, has been shown to sustain ammonia excretion
(DeVries and Wolcott, 1993).
O. quadrata also recycles the urine to produce `P', which can be as
little as 10% of the osmotic strength of the primary urine
(Wolcott and Wolcott, 1985
).
However, the mechanism of NH4+ excretion is quite
different from that of gecarcinids because the concentration in the primary
urine of the ghost crab is extraordinarily high. For example, in O.
quadrata (DeVries and Wolcott,
1993
; DeVries et al.,
1994
) this reaches 116212 mmol l1, and in
O. ceratopthalma and O. cordimanus (under field conditions)
>40 and 27 mmol l1, respectively (S. Morris,
unpublished). The primary urine of Ocypode quadrata is unusually
acidic (pH 5.36±0.21), providing an `acid-trap' for
NH4+. On passage over the gills, the pH is increased (pH
7.01±0.24) and Cl (but not Na+) is
reclaimed, such that the alkalinization of the fluid promotes significant
NH3 volatilization (
71 µl kg1
h1 in control crabs). While pH 7 is not alkaline, the
increase in pH is quite effective. For example, at an ammonia concentration of
116 mmol l1 and pH 5.4 for primary urine
(DeVries & Wolcott, 1993
),
if the pH is increased to pH 7 it is possible to estimate
PNH3 using the pK and solubility for
NH3 provided by Kormanik and Cameron
(1981
) as used by Varley and
Greenaway (1994
). In the
primary urine PNH3=11.6 Pa whereas at pH 7 the
PNH3=460 Pa, which is a 40-fold increase in
potential diffusive gradient. In view of the concomitant increase in the fluid
CO2 concentration and the uptake of Cl, the most
obvious candidate for the net base excretion is transport by an apical
HCO3/Cl exchanger
(DeVries & Wolcott, 1993
).
Reclamation of urinary Na+ appears to be accomplished within the
antennal gland (DeVries et al.,
1994
). These authors (DeVries
et al., 1994
) report high activity of
Na+/K+ATPase in the antennal gland of O.
quadrata for which NH4+ may substitute for
K+ in the basal membrane exchange. Furthermore, apical
Na+/H+ antiporters in the antennal gland may sustain
both Na+ reclamation and acidification of urine to promote
NH4+-trapping (Fig.
8).
|
Study of the more-terrestrial grapsid, Geograpsus grayi, has
revealed further modifications of the
NH3/NH4+ excretory system
(Greenaway and Nakamura, 1991;
Varley and Greenaway, 1994
).
G. grayi is a highly active carnivorous land crab and also
reprocesses the urine to reclaim salts via branchial uptake
(Greenaway and Nakamura, 1991
)
but, unlike Ocypode sp., does not employ ion reclamation within the
antennal gland (Varley and Greenaway,
1994
). Clearly Ocypode and Geograpsus represent
separate radiations in to the terrestrial habitat, but with superficially
analogous ammonia excretion physiology. G. grayi volatilizes
NH3 from the limited volume of `P' within the branchial chamber
and, thereby, increases the effective NH4+ capacity of
the fluid, which may achieve concentrations in excess of 80 mmol
l1 compared with <1 mmol l1 in the
urine (Varley and Greenaway,
1994
). However, G. grayi manages a rate of ammonia
excretion comparable to that of aquatic crabs in water (107220 µmol
kg1 h1;
Greenaway and Nakamura, 1991
;
Varley and Greenaway, 1994
).
Gaseous ammonia contributes
78% of this total excretion in a
discontinuous process over 3 h to 3 days
(Varley and Greenaway, 1994
)
although urine flow is apparently limited, restricting fluid available for `P'
formation. The pH of this fluid (pH 8.07) is higher than that of the hemolymph
(pH 7.667.59) and at the same time the CO2 content (36 mmol
l1) is considerably greater than that of the hemolymph
(13.717.2 mmol l1). Amiloride reduced
NH4+ efflux by 83% in this system and reduced
unidirectional Na+ uptake. Thus, NH3 volatilization is
achieved by raising the fluid pH towards the pK, such that gaseous
NH3 becomes 8% of the total ammonia, creating a diffusive gradient
(PNH3
3.3 Pa) into the convective air
stream. Varley and Greenaway
(1994
) discussed the
difficulties in transporting NH3/NH4+ outward
into this fluid in the absence of `acid-trapping' and proposed a net excretion
reaction
NH4++HCO3
H2O+CO2
+NH3
.
The volatilization of NH3 and CO2, together with
formation of water, all contribute to lowered ionic strength in the
extrabranchial fluid. Again, apical NH4+ transport,
possibly mediated by a putative Rhesus-related ammonia transporter protein,
needs to be investigated in further studies. This mechanism in G.
grayi effectively increases the functional volume of the `P' offering
some escape from the dependency on regular and significant urine flow rate.
Thus, there are significant increases in the amount of ammonia excreted per
unit volume of `P', while retaining the advantages of ammonia excretion,
thereby allowing a more terrestrial habit
(Fig. 9). At the same time, the
system remains potentially limited by the supply of Na+ and
Cl in a lowered supply of urine.
|
The most successful terrestrial animals have abandoned NH3 as an
N-excretory vehicle in favor of urea or purines. The anomuran Birgus
latro is the only identified purinotelic crab
(Greenaway and Morris, 1989)
but is sympatric with several species of gecarcinids that retain
NH3 excretion. Excreting purines allows greater flexibility of
urinary water flow independently of urine reprocessing and salt reclamation,
but significant energetic advantages may accompany ammonotely.
Implications
Decapod crustaceans exhibit a wide variety of ammonia excretion mechanisms
and consequently provide good models for general investigation of nitrogen
excretion. The debate as to whether ammonia is lost via
NH3 diffusion or by NH4+ transport remains
active, but the answer may be both or either depending on circumstance and
species. The ability to move ammonia against its gradient is obviously
essential. There is a clear continuum of increased terrestriality accompanied
by managed and active excretion with lowered water loss. This continuum
represents multiple transitions onto land and is underpinned by phylogenetic
differences. The increased application of molecular and post-genomic
methodologies to the question will reveal, for example, the role of
Rhesus-related proteins and vesicular transport systems in the physical
extrusion of ammonia.
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Acknowledgments |
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Footnotes |
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References |
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