Osmoregulation by gills of euryhaline crabs: Molecular analysis of transporters

American Zoologist, Sep 2001 by Towle, David W, Weihrauch, Dirk

Osmoregulation by Gills of Euryhaline Crabs: Molecular Analysis of Transporters1

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 mamas, including a Na^sup ^ K^sup ^ATPase ax-subunit, a V-type H^sup ^-ATPase B-subunit, a Na^sup ^/H^sup ^ exchanger, a Na^sup ^/ K^sup ^/2Cl^sup -^ 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^sup ^ K^sup ^-ATPase et-subunit and Na^sup ^/H^sup ^ exchanger. The osmoregulatory response to salinity reduction includes enhanced mRNA expression of at least one form of carbonic anhydrase.

INTRODUCTION

Many of the papers in the symposium Osmoregulation: An Integrated Approach are focused at the cellular level, asking questions about mechanisms by which cells regulate their intracellular osmotic condition. At the organismal level, we can ask similar questions about regulation of extracellular osmolytes, focusing on tissues that are specialized for osmolyte uptake, retention, or excretion. This review addresses such an organismal level of osmoregulation, with an emphasis on applications of molecular biology to studies of osmolyte transporters and transport-related enzymes in gills of euryhaline crustaceans.

Among the brachyuran crabs, some species (e.g., the blue crab Callinectes sapidus) are capable of osmoregulating and thriving in freshwater (Mangum and Amende, 1972; Cameron, 1978). Others (e.g., the shore crab Carcinus maenas) are less tolerant of freshwater but nevertheless maintain high extracellular osmolyte concentrations well into brackish water (Siebers et al., 1982). Still others (e.g., the lesser blue crab Callinectes similis) appear less capable of osmoregulation over their entire range (Piller et al., 1995; Guerin and Stickle, 1997) (Fig. 1). The range of osmoregulatory capacities expressed in aquatic crustaceans thus affords the possibility of a comparative physiological approach to the identification of the specific genetic capabilities that contribute to osmoregulatory capacity.

Which organ is most involved in organismal osmoregulation in aquatic crustaceans? The antennal glands of brachyuran crabs are incapable of producing urine that is anisosmotic to the hemolymph (Cameron and Batterton, 1978) and thus cannot be considered as important organs in osmoregulation. Rather, the loss of osmolytes via the urine presents a stressful condition in dilute salinities that must be met by active ion uptake. The crustacean hepatopancreas expresses a variety of transporters (Ahearn et al., 1992), but little attention has been paid to their possible involvement in osmoregulation by euryhaline species. Although the crustacean intestine is known to transport ions (Mantel and Farmer, 1983) and appears to be the major site of water uptake at molt (Neufeld and Cameron, 1994), its overall contribution to osmoregulatory ion uptake is believed to be minimal (Chu, 1987).

The majority of experimental evidence suggests that it is the gills of euryhaline crustaceans that serve as the primary site of osmoregulatory ion transport. Although varying widely in morphology, gills and associated structures of osmoregulators typically contain cell types that are reflective of their transporting capabilities (Taylor and Taylor, 1992). Typical characteristics of these epithelial cells include a greatly elaborated membrane surface, particularly in the basolateral region facing the body fluid. Numerous mitochondria fill such cells, providing the ATP and related phosphagens that power transport processes (Fig. 2). Gill epithelial cells that are believed to function principally in gas exchange are much thinner, with limited membrane elaboration and few mitochondria (Copeland and Fitzjarrell, 1968; Goodman and Cavey, 1990).

Studies of intact animals, isolated gills, gill lamellae, and membrane vesicles have produced a variety of models of osmoregulatory ion transport in euryhaline crustaceans (Lucu, 1990; Towle, 1990; Taylor and Taylor, 1992; Pequeux, 1995; Onken and Riestenpatt, 1998). Nearly universal, however, is the assumption that the major driving force for ion transport across gills is provided by the sodium pump or Na^sup ^ K^sup ^-dependent ATPase (Towle, 1990). In isolated perfused gills of the blue crab, for example, the Na^sup ^ K^sup ^-ATPase inhibitor ouabain blocks more than 50% of the Na uptake from external medium to the hemolymph side (Burnett and Towle, 1990) (Fig. 3). Ouabain also blocks ammonia excretion across isolated gills, supporting the idea that a functional sodium pump is essential for this important process as well (Lucu et al., 1989; Weihrauch et al., 1998).