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Papers by C. Spanings-Pierrot
Journal of Experimental Biology, 2001
SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enh... more SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na++K+-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3′ and 5′ untranslated regions of the mRNA coding for the α-subunit of Na++K+-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The α-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81–83 % identity to those of other arthropods and 74...
Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustace... more Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustacean capable of osmoregulation usually results in a decrease of its Na q and Cl y regulation Ž andror of its osmoregulatory capacity OC: difference between the osmotic pressures of the. hemolymph and of the external medium, at a given salinity. The partial or complete loss of osmoregulatory and ionoregulatory capacity is generally linked to disruptions of the osmotic and ionic regulations. Different causes of these variations include alterations in the structure and ultrastructure of the branchial and excretory organs and changes in Na q ,K q-ATPase activity, ionic fluxes and surface permeability. OC appears therefore as a nonspecific bioindicator. Hyper-OC andror hypo-OC can be measured according to the type of osmoregulation of the species, respectively, in low-salinity Ž media and in seawater or high-salinity media. Provided several precautions are taken control of. salinity and temperature, size, nutritional status, developmental stage, molt stage, use of controls , measurement of OC variations is proposed as a convenient and reliable way to monitor the physiological condition and the effect of stressors in crustaceans.
Journal of Experimental Biology, 2007
Peptides, 2006
Crustacean hyperglycemic hormone X-organ Pericardial organ Brachyura Heterotremata Alternative sp... more Crustacean hyperglycemic hormone X-organ Pericardial organ Brachyura Heterotremata Alternative splicing a b s t r a c t
Journal of Endocrinology, 2006
The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopio... more The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na C /K C -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1a (ef1a) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prl was 4 . 5-fold higher in FW than in SW, whereas pituitary sl was 1 . 8-fold higher in SW-compared with FW-adapted fish. Gill Na C /K C -ATPase specific activity was 2 . 3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6 . 5-fold lower in SW-than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FW euryhaline teleosts.
General and Comparative Endocrinology, 1995
Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused w... more Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was > 5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.
General and Comparative Endocrinology, 2000
Statistical comparisons of experimental data were based on Student's t test, one-way analysis of ... more Statistical comparisons of experimental data were based on Student's t test, one-way analysis of variance Control of Osmoregulation by CHH 343
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2007
Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its ent... more Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na(+)/K(+)-ATPase alpha-subunit, and one putative housekeeping gene, beta-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na(+)/K(+)-ATPase alpha-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). beta-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na(+)/K(+)-ATPase alpha-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 mum), and one in embryos just before hatching (at EI=420 mum), continuing in the freshly hatched juveniles. The Na(+)/K(+)-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.
Archives Of Physiology And Biochemistry, 1995
With the same saline on both sides of the epithelium, a spontaneous inside negative transepitheli... more With the same saline on both sides of the epithelium, a spontaneous inside negative transepithelial potential difference (PD) was measured in perfused posterior gills of the euryhaline crab Pachygrapsus marmoratus acclimated to dilute sea water. The origin of the PD and the transport properties of the epithelium were investigated by ionic substitutions and by application of inhibitors. Diffusion of Na+ and Cl- ions at the apical side contributes to the establishment of the PD. Sodium cyanide (10(-2) M) added to the perfusion and incubation media almost completely inhibited the transepithelial PD and considerably decreased the Na+ influx. Internally perfused ouabain (5.10(-3) M) halved the PD and the Na+ influx but had no effect on the Na+ efflux. Externally applied amiloride (10(-3) M) also reduced the Na+ influx by 27%. All inhibitions were concentration-dependent. From these results, it has been concluded that the transepithelial PD and Na+ influx are, at least partly, generated by active, metabolic energy-requiring processes. The effect of ouabain supports the existence of a Na+/K+ exchange mechanism linked to the presence of Na(+)-K+ ATPase in the basolateral membrane of posterior gills. The effect of amiloride is discussed.
Archives Of Physiology And Biochemistry, 1995
This paper reports the adaptation of a branchial perfusion technique to the gills of the small hy... more This paper reports the adaptation of a branchial perfusion technique to the gills of the small hyper-hyporegulating crab, Pachygrapsus marmoratus. The physiological quality of the preparation has been established and preliminary measurements of the transepithelial potential difference (PD) and sodium fluxes were reported. A clear-cut distinction has to be made between anterior and posterior gills. With symmetrical bathing conditions (same saline on both sides of the epithelium), a significant transepithelial PD was measured only in posterior gills isolated from crabs acclimated to dilute sea water. This hemolymph-side negative PD is shown to require biological energy and to be sensitive to ouabain. Na+ influx was larger than efflux, indicating the occurrence of a net inward of Na+. The active nature of the Na+ influx was established.
Aquaculture, 2000
Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustace... more Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustacean capable of osmoregulation usually results in a decrease of its Na q and Cl y regulation Ž andror of its osmoregulatory capacity OC: difference between the osmotic pressures of the . hemolymph and of the external medium, at a given salinity . The partial or complete loss of osmoregulatory and ionoregulatory capacity is generally linked to disruptions of the osmotic and ionic regulations. Different causes of these variations include alterations in the structure and ultrastructure of the branchial and excretory organs and changes in Na q ,K q -ATPase activity, ionic fluxes and surface permeability.
The Journal of experimental biology, 2001
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced N... more Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other ...
Journal of Experimental Biology, 2001
SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enh... more SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na++K+-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3′ and 5′ untranslated regions of the mRNA coding for the α-subunit of Na++K+-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The α-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81–83 % identity to those of other arthropods and 74...
Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustace... more Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustacean capable of osmoregulation usually results in a decrease of its Na q and Cl y regulation Ž andror of its osmoregulatory capacity OC: difference between the osmotic pressures of the. hemolymph and of the external medium, at a given salinity. The partial or complete loss of osmoregulatory and ionoregulatory capacity is generally linked to disruptions of the osmotic and ionic regulations. Different causes of these variations include alterations in the structure and ultrastructure of the branchial and excretory organs and changes in Na q ,K q-ATPase activity, ionic fluxes and surface permeability. OC appears therefore as a nonspecific bioindicator. Hyper-OC andror hypo-OC can be measured according to the type of osmoregulation of the species, respectively, in low-salinity Ž media and in seawater or high-salinity media. Provided several precautions are taken control of. salinity and temperature, size, nutritional status, developmental stage, molt stage, use of controls , measurement of OC variations is proposed as a convenient and reliable way to monitor the physiological condition and the effect of stressors in crustaceans.
Journal of Experimental Biology, 2007
Peptides, 2006
Crustacean hyperglycemic hormone X-organ Pericardial organ Brachyura Heterotremata Alternative sp... more Crustacean hyperglycemic hormone X-organ Pericardial organ Brachyura Heterotremata Alternative splicing a b s t r a c t
Journal of Endocrinology, 2006
The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopio... more The mRNA expression of pituitary prolactin (prl), growth hormone (gh), somatolactin (sl), proopiomelanocortin (pomc), and gonadotropins (gthI and gthII) was quantified by real-time PCR, in sea bass, Dicentrarchus labrax, adapted for 1 month to seawater (SW) or freshwater (FW). In addition, IGF-I (igfI) mRNA expression in liver and branchial Na C /K C -ATPase activity were determined. L17 ribosomal protein (rpL17) and elongation factor 1a (ef1a) were validated as reference genes in real-time PCR in the experimental context. The real-time PCR assays were validated for the different hormone genes considered. Expression of pituitary pomc, gthI, gthII, gh, and liver igfI was not significantly different between FW and SW fish. Pituitary prl was 4 . 5-fold higher in FW than in SW, whereas pituitary sl was 1 . 8-fold higher in SW-compared with FW-adapted fish. Gill Na C /K C -ATPase specific activity was 2 . 3-fold higher in FW sea bass compared with SW fish. Plasma cortisol levels were 6 . 5-fold lower in SW-than in FW-adapted specimens. The results are discussed in relation to the osmoregulatory strategy of this euryhaline SW species, which displays features that do not fit present models based on salmonids and FW euryhaline teleosts.
General and Comparative Endocrinology, 1995
Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused w... more Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was > 5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.
General and Comparative Endocrinology, 2000
Statistical comparisons of experimental data were based on Student's t test, one-way analysis of ... more Statistical comparisons of experimental data were based on Student's t test, one-way analysis of variance Control of Osmoregulation by CHH 343
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2007
Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its ent... more Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na(+)/K(+)-ATPase alpha-subunit, and one putative housekeeping gene, beta-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na(+)/K(+)-ATPase alpha-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). beta-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na(+)/K(+)-ATPase alpha-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 mum), and one in embryos just before hatching (at EI=420 mum), continuing in the freshly hatched juveniles. The Na(+)/K(+)-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.
Archives Of Physiology And Biochemistry, 1995
With the same saline on both sides of the epithelium, a spontaneous inside negative transepitheli... more With the same saline on both sides of the epithelium, a spontaneous inside negative transepithelial potential difference (PD) was measured in perfused posterior gills of the euryhaline crab Pachygrapsus marmoratus acclimated to dilute sea water. The origin of the PD and the transport properties of the epithelium were investigated by ionic substitutions and by application of inhibitors. Diffusion of Na+ and Cl- ions at the apical side contributes to the establishment of the PD. Sodium cyanide (10(-2) M) added to the perfusion and incubation media almost completely inhibited the transepithelial PD and considerably decreased the Na+ influx. Internally perfused ouabain (5.10(-3) M) halved the PD and the Na+ influx but had no effect on the Na+ efflux. Externally applied amiloride (10(-3) M) also reduced the Na+ influx by 27%. All inhibitions were concentration-dependent. From these results, it has been concluded that the transepithelial PD and Na+ influx are, at least partly, generated by active, metabolic energy-requiring processes. The effect of ouabain supports the existence of a Na+/K+ exchange mechanism linked to the presence of Na(+)-K+ ATPase in the basolateral membrane of posterior gills. The effect of amiloride is discussed.
Archives Of Physiology And Biochemistry, 1995
This paper reports the adaptation of a branchial perfusion technique to the gills of the small hy... more This paper reports the adaptation of a branchial perfusion technique to the gills of the small hyper-hyporegulating crab, Pachygrapsus marmoratus. The physiological quality of the preparation has been established and preliminary measurements of the transepithelial potential difference (PD) and sodium fluxes were reported. A clear-cut distinction has to be made between anterior and posterior gills. With symmetrical bathing conditions (same saline on both sides of the epithelium), a significant transepithelial PD was measured only in posterior gills isolated from crabs acclimated to dilute sea water. This hemolymph-side negative PD is shown to require biological energy and to be sensitive to ouabain. Na+ influx was larger than efflux, indicating the occurrence of a net inward of Na+. The active nature of the Na+ influx was established.
Aquaculture, 2000
Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustace... more Exposure to water-borne pollutants, environmental stressors and pathological agents of a crustacean capable of osmoregulation usually results in a decrease of its Na q and Cl y regulation Ž andror of its osmoregulatory capacity OC: difference between the osmotic pressures of the . hemolymph and of the external medium, at a given salinity . The partial or complete loss of osmoregulatory and ionoregulatory capacity is generally linked to disruptions of the osmotic and ionic regulations. Different causes of these variations include alterations in the structure and ultrastructure of the branchial and excretory organs and changes in Na q ,K q -ATPase activity, ionic fluxes and surface permeability.
The Journal of experimental biology, 2001
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced N... more Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other ...