Dirk Weihrauch

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

Ammonia excretion in aquatic and terrestrial crabs.

SUMMARY The excretory transport of toxic ammonia across epithelia is not fully understood. This review presents data combined with models of ammonia excretion derived from studies on decapod crabs, with a view to providing new impetus to investigation of this essential issue. The majority of crabs preserve ammonotely regardless of their habitat, which varies from extreme hypersaline to freshwater aquatic environments, and ranges from transient air exposure to obligate air breathing. Important components in the excretory process are the Na+/K+(NH4+)-ATPase and other membrane-bound transport proteins identified in many species, an exocytotic ammonia excretion mechanism thought to function in gills of aquatic crabs such as Carcinus maenas, and gaseous ammonia release found in terrestrial crabs, such as Geograpsus grayi and Ocypode quadrata. In addition, this review presents evidence for a crustacean Rhesus-like protein that shows high homology to the human Rhesus-like ammonia transporter both in its amino acid sequence and in its predicted secondary structure.

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas☆

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzymes role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.

Osmoregulation by Gills of Euryhaline Crabs: Molecular Analysis of Transporters

SYNOPSIS. The physiological mechanisms by which aquatic animals regulate the osmoconcentration of their body fluids remain unclear despite many excellent studies of tissue and cell function. This review summarizes the current status of an ongoing molecular biological approach to investigating transporters and transportrelated enzymes in ion-transporting gills of osmoregulating crustaceans. We have identified cDNAs coding for six candidate proteins in gills of the blue crab Callinectes sapidus and the green shore crab Carcinus maenas, including a Na 1 KATPase a-subunit, a V-type H-ATPase B-subunit, a Na/H exchanger, a Na/ K/2Cl cotransporter, two isoforms of carbonic anhydrase, and arginine kinase. Although our account is far from complete, examination of mRNA abundance by quantitative reverse transcription/polymerase chain reaction (RT/PCR) has identified candidates that are preferentially expressed in gill epithelium, including the Na 1 K-ATPase a-subunit and Na/H exchanger. The osmoregulatory response to salinity reduction includes enhanced mRNA expression of at least one form of carbonic anhydrase.

Potential of active excretion of ammonia in three different haline species of crabs

Abstract Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted 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 μmol · l−1 NH4Cl in the perfusion saline and concentrations of NH4Cl in the bath that were stepwise increased from 0 to 4000 μmol · l−1 allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different 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 μmol · l−1 NH+4) through the gill epithelium against ambient concentrations of up to 800 μmol · 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 influx at very high external concentrations of up to 4000 μmol · 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 increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences 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 influx 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 influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm−2) which may reduce passive NH+4 influxes at high ambient levels.

Osmoregulation and Excretion

The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and their integration in maintaining whole body water and ion homeostasis. The focus is on recent studies of adaptations and newly discovered mechanisms of acclimatization during transitions of animals between different osmotic environments. Special attention is paid to hypotheses about the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with accounts on experimental approaches and methods applied in the studies. It is demonstrated how knowledge in these areas of comparative physiology has expanded considerably during the last two decades, bridging seminal classical works with studies based on new approaches at all levels of anatomical and functional organization. A number of as yet partially unanswered questions are emphasized, some of which are about how water and solute exchange mechanisms at lower levels are integrated for regulating whole body extracellular water volume and ion homeostasis of animals in their natural habitats.

Induction of branchial ion transporter mRNA expression during acclimation to salinity change in the euryhaline crab Chasmagnathus granulatus.

SUMMARY Using quantitative real-time PCR, the expression of mRNAs encoding three transport-related proteins and one putative housekeeping protein was analyzed in anterior and posterior gills of the euryhaline crab Chasmagnathus granulatus following transfer from isosmotic conditions (30‰ salinity) to either dilute (2‰) or concentrated (45‰) seawater. Modest changes were observed in the abundance of mRNAs encoding the housekeeping protein arginine kinase and the vacuolar-type H+-ATPase B-subunit, both of which were highly expressed under all conditions. By contrast, the expression of Na+/K+-ATPaseα -subunit mRNA and Na+/K+/2Cl- cotransporter mRNA was strongly responsive to external salinity. During acclimation to dilute seawater, cotransporter mRNA increased 10-20-fold in posterior gills within the first 24 h while Na+/K+-ATPase α-subunit mRNA increased 35-55-fold. During acclimation to concentrated seawater, cotransporter mRNA increased 60-fold by 96 h and Na+/K+-ATPase α-subunit increased approximately 25-fold in posterior gills. Our results indicate a complex pattern of transcriptional regulation dependent upon the direction of salinity change and the developmental background of the gills.

ACTIVE EXCRETION OF AMMONIA ACROSS THE GILLS OF THE SHORE CRAB CARCINUS MAENAS AND ITS RELATION TO OSMOREGULATORY ION UPTAKE

Abstract The mechanism of transbranchial excretion of total ammonia of brackish-water acclimated shore crabs, Carcinus maenas was examined using isolated, perfused gills. Applying physiological gradients of NH4Cl (100–200 μmol · l−1) directed from the haemolymph space to the bath showed that the efflux of total ammonia consisted of two components. The saturable component (excretion of NH4+) greatly exceeded the linear component (diffusion of NH3). When an outwardly directed gradient (200 μmol · l−1) was applied, total ammonia in the perfusate was reduced by more than 50% during a single passage of saline through the gill. Effluxes of ammonia along the gradient were sensitive to basolateral dinitrophenol, ouabain, and Cs+ and to apical amiloride. Acetazolamide (1 mmol · l−1 basolateral) or Cl−-free conditions had no substantial effects on ammonia flux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory NaCl uptake. When an inwardly directed gradient (200 μmol · l−1) was employed, influx rates were about 10-fold smaller and unaffected by basolateral ouabain (5 mmol · l−1) or dinitrophenol (0.5 mmol · l−1). Under symmetrical conditions (100 μmol · l−1 NH4Cl on both sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during the experiment and against the gradient of the partial pressure of NH3. The active excretion rate was reduced to 7% of controls by basolateral dinitrophenol (0.5 mmol · l−1), to 44% by basolateral ouabain (5 mmol · l−1), to 46% by Na+-free conditions and to 42% by basolateral Cs+ (10 mmol · l−1), indicating basolateral membrane transport of NH4+ via the Na+/K+-ATPase and K+-channels and a second active, apically located, Na+ independent transport mechanism of NH4+. Anterior gills, which are less capable of active ion uptake than posterior gills, exhibited even increased rates of active excretion of ammonia. We conclude that, under physiological conditions, branchial excretion of ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an inwardly directed gradient, if necessary.

Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae)

SUMMARY The present investigation examined the microanatomy and mRNA expression and activity of ion-motive ATPases, in anterior and posterior gills of a South American, true freshwater crab, Dilocarcinus pagei. Like diadromous crabs, the anterior gills of this hololimnetic trichodactylid exhibit a highly attenuated (2–5 μm), symmetrical epithelium on both lamellar surfaces. In sharp contrast, the posterior gill lamellar epithelia are markedly asymmetrical. Their proximal side consists of thick (18–20μ m) cells, displaying features typical of a transporting epithelium, while the distal epithelium is thin (3–10 μm) and formed entirely by apical pillar cell flanges. Both anterior and posterior gills express Na+/K+- and V-ATPases. Phylogenetic analysis of partial cDNA sequences for the Na+/K+-ATPase α-subunit and V-ATPase B-subunit among various crab species confirmed the previous classification and grouping of D. pagei based on morphological criteria. Semi-quantitative RT-PCR clearly showed that mRNA for both ion pump subunits is more intensely expressed in posterior gills. Na+/K+-ATPase activity in the posterior gills was nearly fourfold that of anterior gills, while V-ATPase and F-ATPase activities did not differ. A negative short-circuit current (Isc) was measured using the distal side of split, posterior gill lamellae, mounted in a modified Ussing chamber and perfused symmetrically with identical hemolymph-like salines. Although hemolymph-side ouabain did not affect this current, concanamycin significantly reduced Isc without altering preparation conductance, suggesting V-ATPase-driven Cl– absorption on the distal side of the posterior gill lamellae, as known to occur in diadromous crabs adapted to freshwater. These findings suggest that active Na+ uptake predominates across the thick proximal epithelium, and Cl– uptake across the thin, distal epithelium of the posterior gill lamellae.

Gill-specific transcriptional regulation ofNa+/K+-ATPase α-subunit in the euryhaline shorecrab Pachygrapsus marmoratus: sequence variants and promoterstructure

SUMMARY The sodium pump (Na+/K+-ATPase) has been implicated in osmoregulatory ion transport in many aquatic animals. In the euryhaline hyper–hypoosmoregulating shore crab Pachygrapsus marmoratus, induction of Na+/K+-ATPase α-subunit mRNA varies between gills in response to osmotic stress. Following transfer of crabs from normal seawater (36‰ salinity) to diluted seawater (10‰), a condition in which gills exhibit net ion uptake, α-subunit mRNA expression is upregulated in all tested gills, albeit with differing time courses. By contrast, following transfer from seawater to hypertonic (45‰) seawater, a condition in which the animal is excreting ions,α -subunit mRNA is induced primarily in gill no. 7 (nine in total), suggesting that this gill may be associated specifically with ion excretion in P. marmoratus. Full-length sequencing of α-subunit cDNA revealed the existence of two isoforms differing only in the inclusion of an 81-nucleotide segment within the N-terminal open reading frame of the long (D) form in comparison to the short (C) form. The 81-nucleotide segment encodes a 14-3-3 protein binding site that may facilitate movement of the α-subunit protein between intracellular compartments and the plasma membrane. mRNA expression of the two forms followed similar patterns upon salinity transfer. Genomic DNA sequencing of the putative promoter region of the α-subunit gene demonstrated a spectrum of predicted transcription factor binding sites that are likely associated with the complex expression pattern observed among gills following osmotic stress.