Carlos M. Luquet

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 NaCl absorption across posterior gills of hyperosmoregulating Chasmagnathus granulatus.

SUMMARY Split lamellae of posterior gills of Chasmagnathus granulatus adapted to 2.5‰ salinity were mounted in a modified Ussing chamber. With NaCl-saline on both sides of the preparation a transepithelial voltage (Vte) of 4.1±0.5 mV (outside positive) was measured. After voltage-clamping, the negative short-circuit current (Isc) amounted to -142±21 μA cm-2 at a conductance (Gte) of 44±5 mS cm-2. Substitution of either chloride (by nitrate) or sodium (by choline) on both sides of split gill lamellae significantly reduced Isc (by 70-80%) and Gte (by 30-50%). External CsCl (but not BaCl2 or furosemide) inhibited the negative Isc without affecting Gte. Addition of ouabain, BaCl2 or diphenylamine-2-carboxylate to the internal bath inhibited Isc at unchanged Gte. Internal acetazolamide did not affect Isc or Gte across split gill lamellae. Unidirectional Na+ influx across isolated and perfused posterior gills, however, was reduced by internal acetazolamide by approximately 20% at constant Vte. The results suggest that posterior gills of hyperosmoregulating C. granulatus display a high conductance epithelium that actively absorbs NaCl in a coupled way by an electrogenic mechanism similar to that seen in the thick ascending limb of Henles loop and, to a minor degree, by an electroneutral mechanism, presumably via apical Na+/H+- and Cl-/HCO3--antiports.

Acid–Base Balance and Ionic Regulation During Emersion in the Estuarine Intertidal Crab Chasmagnathus granulata Dana (Decapoda Grapsidae)

Abstract Haemolymphatic P O 2 , P CO 2 , and pH, and ionic concentrations of haemolymph and branchial chamber water were measured in submerged and emersed crabs. Hypercapnia and respiratory acidosis were recorded after 15 min of air exposure, then P CO 2 remained constant and pH was compensated. Sodium concentration increased in haemolymph and decreased in branchial water. The difference between haemolymph Na + and Cl − increased dramatically. Acid–base balance is restored by two mechanisms: a) increase of the P CO 2 gradient across the gills and consequently the rate of CO 2 excretion; b) the increase in the strong ion difference, caused by branchial sodium uptake, compensates the hypercapnic acidosis.

GILL MORPHOLOGY OF THE INTERTIDAL ESTUARINE CRAB CHASMAGNATHUS GRANULATUS DANA, 1851 (DECAPODA, GRAPSIDAE) IN RELATION TO HABITAT AND RESPIRATORY HABITS

[Histological and morphometric analyses were performed on the gills of the semiterrestrial estuarine crab Chasmagnathus granulatus. Three different epithelia were recognized: (1) A thin epithelium, 2:42 ± 0:33 mum thick, which lines the whole lamellae in the three anterior gills and was assigned to respiratory functions. (2) A thick cuboidal epithelium, 6-12.5 mum thick, covering part of gills 4 and 5 and the better part of the three most posterior gills. This tissue seems to be involved in ion-regulation, since it is located in the same zones which are darkly stained with silver nitrate and possess large numbers of mitochondria, closely associated to basolateral interdigitations and abundant infoldings of the apical membrane. (3) An attenuated epithelium, 0.5-1.5 mum thick, bordering the marginal channels of all the gills. This tissue is clearly of a respiratory type and probably plays an important role during air breathing when the branchial water stores become reduced by evaporation, and the consequent gill collapse impairs both ventilation and perfusion of the central part of the lamellae. During exposure to humid air, C. granulatus is able to maintain its branchial chambers almost completely filled with water, thus keeping its gills functional in spite of little mechanical support. These adaptations for maintaining gill respiration in air allow C. granulatus to sustain high metabolic rates during emergence, with little increase in venous partial pressure of carbon dioxide. Se realizaron estudios histologicos y morfometricos de las branquias del cangrejo semiterrestre estuarial Chasmagnathus granulatus. Se identificaron tres tipos diferentes de epitelio: (1) Un tejido delgado, de 2:42 ± 0:33 mum de altura, al cual se le atribuyen funciones respiratorias y tapiza toda la superficie de las tres branquias anteriores y parte de las posteriores. (2) Un epitelio cuboide alto de 6-12.5 mum, que cubre parte de las branquias 4 y 5 y la mayor parte de las tres branquias mas posteriores. Este tejido puede considerarse ionorregulatorio, ya que se ubica en las mismas areas que se tinen intensamente con nitrato de plata y presenta gran cantidad de mitocondrias estrechamente ligadas a interdigitaciones de la membrana basolateral y profundos repliegues de la membrana apical. (3) Un epitelio atenuado de 0.5-1.5 mum, que bordea el canal marginal en las laminillas de todas las branquias y tiene un aspecto claramente respiratorio. Este tejido posiblemente cumple un papel importante en la respiracion aerea, cuando el volumen de agua retenida en las camaras branquiales disminuye por evaporacion y consecuentemente, las laminillas de las branquias se adhieren entre si, lo cual dificulta la ventilacion y la perfusion de su porcion central. Cuando respira aire humedo C. granulatus es capaz de mantener sus camaras branquiales practicamente llenas de agua, esto permite mantener funcionales a sus branquias, que son relativamente grandes y carecen de soporte mecanico para la respiracion aerea. Estas adaptaciones para mantener la respiracion branquial en aire permiten a esta especie mantener una alta tasa metabolica durante la emersion manteniendo bajos niveles de presion de dioxido de carbono., Histological and morphometric analyses were performed on the gills of the semiterrestrial estuarine crab Chasmagnathus granulatus. Three different epithelia were recognized: (1) A thin epithelium, 2:42 ± 0:33 mum thick, which lines the whole lamellae in the three anterior gills and was assigned to respiratory functions. (2) A thick cuboidal epithelium, 6-12.5 mum thick, covering part of gills 4 and 5 and the better part of the three most posterior gills. This tissue seems to be involved in ion-regulation, since it is located in the same zones which are darkly stained with silver nitrate and possess large numbers of mitochondria, closely associated to basolateral interdigitations and abundant infoldings of the apical membrane. (3) An attenuated epithelium, 0.5-1.5 mum thick, bordering the marginal channels of all the gills. This tissue is clearly of a respiratory type and probably plays an important role during air breathing when the branchial water stores become reduced by evaporation, and the consequent gill collapse impairs both ventilation and perfusion of the central part of the lamellae. During exposure to humid air, C. granulatus is able to maintain its branchial chambers almost completely filled with water, thus keeping its gills functional in spite of little mechanical support. These adaptations for maintaining gill respiration in air allow C. granulatus to sustain high metabolic rates during emergence, with little increase in venous partial pressure of carbon dioxide. Se realizaron estudios histologicos y morfometricos de las branquias del cangrejo semiterrestre estuarial Chasmagnathus granulatus. Se identificaron tres tipos diferentes de epitelio: (1) Un tejido delgado, de 2:42 ± 0:33 mum de altura, al cual se le atribuyen funciones respiratorias y tapiza toda la superficie de las tres branquias anteriores y parte de las posteriores. (2) Un epitelio cuboide alto de 6-12.5 mum, que cubre parte de las branquias 4 y 5 y la mayor parte de las tres branquias mas posteriores. Este tejido puede considerarse ionorregulatorio, ya que se ubica en las mismas areas que se tinen intensamente con nitrato de plata y presenta gran cantidad de mitocondrias estrechamente ligadas a interdigitaciones de la membrana basolateral y profundos repliegues de la membrana apical. (3) Un epitelio atenuado de 0.5-1.5 mum, que bordea el canal marginal en las laminillas de todas las branquias y tiene un aspecto claramente respiratorio. Este tejido posiblemente cumple un papel importante en la respiracion aerea, cuando el volumen de agua retenida en las camaras branquiales disminuye por evaporacion y consecuentemente, las laminillas de las branquias se adhieren entre si, lo cual dificulta la ventilacion y la perfusion de su porcion central. Cuando respira aire humedo C. granulatus es capaz de mantener sus camaras branquiales practicamente llenas de agua, esto permite mantener funcionales a sus branquias, que son relativamente grandes y carecen de soporte mecanico para la respiracion aerea. Estas adaptaciones para mantener la respiracion branquial en aire permiten a esta especie mantener una alta tasa metabolica durante la emersion manteniendo bajos niveles de presion de dioxido de carbono.]

Regulation of ion transport by pH and [HCO3−] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.

Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations.

SUMMARY In the present study, the influence of short-term osmotic variations on some electrophysiological properties related to NaCl absorption across posterior gills of Chasmagnathus granulatus was investigated. The transepithelial potential difference (Vte) of isolated and perfused gills increased significantly when hyposmotic saline (699 mosmol l-1) was used instead of isosmotic solution (1045 mosmol l-1). A reduction of the concentration of Na+ or Cl- at constant osmolarity did not produce any change in Vte. Transepithelial short-circuit current (Isc) and conductance (Gte), measured with split gill lamellae mounted in a modified Ussing chamber, also increased after changing to hyposmotic salines (Isc: from -89.0±40.8 μA cm-2 to -179.3±37.0 μA cm-2; Gte: from 40.5±16.9 mS cm-2 to 47.3±15.8 mS cm-2). The observed effects of reduced osmolarity were fast, reversible and gradually dependent on the magnitude of the osmotic variation. The activity of the Na+/K+-ATPase increased significantly after perfusion with hyposmotic saline, from 18.73±6.35 μmol Pi h-1 mg-1 to 41.84±14.54 μmol Pi h-1 mg-1. Theophylline maintained part of the elevated Vte induced by hyposmotic saline, suggesting that an increased cellular cyclic AMP level is involved in the response to reduced osmolarity. In summary, the results indicate that the hemolymph osmolarity regulates active transbranchial NaCl absorption by modulating the activity of the basolateral Na+/K+-ATPase and by changing a conductive pathway, probably at the apical membrane.

Salinity-induced changes in the fine structure of the gills of the semiterrestrial estuarian crab, Uca uruguayensis (Nobili, 1901) (Decapoda, Ocypodidae).

The posterior gills of Uca uruguayensis are mostly lined with a thick tissue which presents the characteristics of a typical salt-transporting epithelium. Electron microscope analysis of gill tissue from crabs acclimated to both low (2.5 per thousand) and high (44 per thousand) salinity showed significant development of the basolateral membrane interdigitations with numerous mitochondria and conspicuous apical membrane infoldings. In high-salinity acclimated crabs, the basolateral interdigitations extended to the apical membrane. Under these conditions, apical infoldings were expanded laterally (forming wide subcuticular spaces), while the apical infoldings of low-salinity adapted animals appeared as regular leaflets. Septate desmosomes were also much more developed in low-salinity exposed animals than in those kept under high-salinity conditions. These morphological observations were analyzed for correlation with the currently-accepted ion hyporegulation model for crustaceans, which is mainly based on transcellular sodium flow. In this study, we propose an ion hyporegulation model involving apical paracellular sodium flux.

Aglomerularism in Harpagifer bispinis: a subantarctic notothenioid fish living at reduced salinity

We investigated the renal morphology, histology and ultrastructure of Harpagifer bispinis, as a first step toward understanding the morpho-functional basis of its adaptation to potentially freezing brackish seawater. Fish were separated into two groups of ten individuals each, and acclimated to 2‰ and 38‰ salinity. A study of complete serial sections of the kidney revealed that the nephrons were aglomerular. At the highly convoluted proximal segment two different regions were evident, a feature that has not been previously reported for other aglomerular species. In electron photomicrographs we distinguished light and dark cells in the proximal tubule epithelium, with highly infolded basolateral membranes and closely associated mitochondria. The dark cells also had a large number of mitochondria in the apical region. The intercellular spaces at the epithelium of the proximal tubule were larger in fish acclimated at 2‰ salinity, a modification that might facilitate urine secretion, thus contributing to the survival of an aglomerular fish in a hyposmotic medium.

Inducing the alternative oxidase forms part of the molecular strategy of anoxic survival in freshwater bivalves

Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O2/L), hypoxia (2 mg O2/L), and normoxia (9 mg O2/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

Ultrastructural and Biochemical Studies of the Branchiostegite of the Bimodal Breathing Crab Chasmagnathus Granulata Dana, 1851

This study provides histologic and ultrastructural evidence of the capacity of Chasmagnathus granulata to perform aerial gas exchange across the lining of branchiostegites. Although the lungs of C. granulata seem to be suitable organs for aerial oxygen uptake, the low levels of carbonic anhydrase activity compared with the gills, indicate limited capacity to excrete carbon dioxide directly to air, resulting in a limiting factor for the crab to invade land.