RESUMEN
Giant clams conduct light-enhanced shell formation, which requires the increased transport of Ca2+ and inorganic carbon (Ci) from the hemolymph through the shell-facing epithelium of the whitish inner mantle to the extrapallial fluid where CaCO3 deposition occurs. The major form of Ci in the hemolymph is HCO3-, but the mechanisms of HCO3- transport through the basolateral and apical membranes of the shell-facing epithelial cells remain unknown. This study aimed to clone from the inner mantle of Tridacna squamosa the complete coding cDNA sequences of electrogenic Na+-HCO3-cotransporter 1 homolog (NBCe1-like-b) and electrogenic Na+-HCO3-cotransporter 2 homolog (NBCe2-like). NBCe1-like-b comprised 3360 bp, encoding a 125.7 kDa protein with 1119 amino acids. NBCe1-like-b was slightly different from NBCe1-like-a of the ctenidium reported elsewhere, as it had a serine residue (Ser1025), which might undergo phosphorylation leading to the transport of Na+: HCO3- at a ratio of 1: 2 into the cell. NBCe1-like-b was localized at the basolateral membrane of the shell-facing epithelial cells, and its gene and protein expression levels increased significantly in the inner mantle during illumination, indicating a role in the light-enhanced uptake of HCO3- from the hemolymph. The sequence of NBCe2-like obtained from the inner mantle was identical to that reported previously for the outer mantle. In the inner mantle, NBCe2-like had an apical localization in the shell-facing epithelial cells, and its protein abundance was upregulated during illumination. Hence, NBCe2-like might take part in the light-enhanced transport of HCO3- through the apical membrane of these cells into the extrapallial fluid.
Asunto(s)
Bicarbonatos , Bivalvos , Animales , Bicarbonatos/metabolismo , Transporte Biológico , Bivalvos/fisiología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismoRESUMEN
Giant clams harbor coccoid Symbiodiniaceae dinoflagellates that are phototrophic. These dinoflagellates generally include multiple phylotypes (species) of Symbiodinium, Cladocopium, and Durusdinium in disparate proportions depending on the environmental conditions. The coccoid symbionts can share photosynthate with the clam host, which in return supply them with nutrients containing inorganic carbon, nitrogen and phosphorus. Symbionts can recycle nitrogen by absorbing and assimilating the endogenous ammonia produced by the host. This study aimed to use the transcript levels of ammonia transporter 2 (AMT2) in Symbiodinium (Symb-AMT2), Cladocopium (Clad-AMT2) and Durusdinium (Duru-AMT2) as molecular indicators to estimate the potential of ammonia transport in these three genera of Symbiodiniaceae dinoflagellates in different organs of the fluted giant clam, Tridacna squamosa, obtained from Vietnam. We also determined the transcript levels of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcII) and nitrate transporter 2 (NRT2) in Symbiodinium (Symb-rbcII; Symb-NRT2), Cladocopium (Clad-rbcII; Clad-NRT2) and Durusdinium (Duru-rbcII; Duru-NRT2), in order to examine the potential of ammonia transport with reference to the potentials of phototrophy or NO3- uptake independent of the quantities and proportion of these Symbiodiniaceae phylotypes. Our results indicated for the first time that phylotypes of Symbiodinium and Cladocopium could have different potentials of ammonia transport, and that phylotypes of Symbiodinium might have higher potential of NO3- transport than ammonia transport. They also suggested that Symbiodiniaceae phylotypes residing in different organs of T. squamosa could have disparate potentials of ammonia transport, alluding to the functional diversity among phylotypes of coccoid Symbiodinium, Cladocopium, and Durusdinium.
Asunto(s)
Antozoos , Bivalvos , Dinoflagelados , Amoníaco/metabolismo , Animales , Antozoos/metabolismo , Bivalvos/metabolismo , Dinoflagelados/fisiología , Proteínas de Transporte de Membrana , Nitrógeno , SimbiosisRESUMEN
African lungfishes are obligatory air-breathers with exceptionally high environmental ammonia tolerance. They can lower the pH of the external medium during exposure to ammonia-loading conditions. This study aimed to demonstrate the possible involvement of branchial vacuolar-type H+-ATPase (Vha) in the ammonia-induced acidification of the external medium by the West African lungfish, Protopterus annectens, and to examine whether its capacity to acidify the medium could be augmented after exposure to 100 mmol l-1 NH4Cl for six days. Two full coding cDNA sequences of Vha subunit B (atp6v1b), atp6v1b1 and atp6v1b2, were obtained from the internal gills of P. annectens. The sequence of atp6v1b1 comprised 1548 bp, encoding 515 amino acids (57.4 kDa), while that of atp6v1b2 comprised 1536 bp, encoding 511 amino acids (56.6 kDa). Using a custom-made antibody reactive to both isoforms, immunofluorescence microscopy revealed the collective localization of Atp6v1b (atp6v1b1 and atp6v1b2) at the apical or the basolateral membrane of two different types of branchial Na+/K+-ATPase-immunoreactive ionocyte. The ionocytes labelled apically with Atp6v1b presumably expressed Atp6v1b1 containing a PDZ-binding domain, indicating that the apical Vha was positioned to transport H+ to the external medium. The expression of Atp6v1b was regulated post-transcriptionally, as the protein abundance of Atp6v1b and Vha activity increased significantly in the gills of fish exposed to 100 mmol l-1 NH4Cl for six days. Correspondingly, the fish exposed to ammonia had a greater capacity to acidify the external medium, presumably to decrease the ratio of [NH3] to [NH4+] in order to reduce the influx of exogenous NH3.
Asunto(s)
Amoníaco , ATPasas de Translocación de Protón Vacuolares , Aminoácidos/metabolismo , Amoníaco/metabolismo , Animales , Peces/fisiología , Branquias/metabolismo , Concentración de Iones de Hidrógeno , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Giant clams perform light-enhanced shell formation (calcification) and therefore need to increase the uptake of exogenous Ca2+ during illumination. The ctenidium of the fluted giant clam, Tridacna squamosa, is involved in light-enhanced Ca2+ uptake. It expresses the pore-forming voltage-gated calcium channel (VGCC) subunit alpha 1 (CACNA1) in the apical membrane of the epithelial cells, and the protein expression level of CACNA1 is upregulated in the ctenidium during illumination. This study aimed to elucidate the mechanism involved in the transport of the absorbed Ca2+ across the basolateral membrane of the ctenidial epithelial cells into the hemolymph. We obtained a homolog of Na+/Ca2+exchanger 1 (NCX1-like) from the ctenidium of T. squamosa, which comprised 2418 bp, encoding a protein of 806 amino acids (88.9 kDa). NCX1-like had a basolateral localization in the epithelial cells of the ctenidial filaments and tertiary water channels. Illumination resulted in significant increases in the transcript and protein levels of NCX1-like/NCX1-like in the ctenidium. Hence, NCX1-like could operate in conjunction with VGCC to increase the transport of Ca2+ from the ambient seawater into the hemolymph during illumination. Illumination also resulted in the upregulation of the gene and protein expression levels of Na+/K+-ATPase (NKA) α-subunit (NKAα/NKAα) in the ctenidium of T. squamosa. As light-enhanced extrusion of Ca2+ into the hemolymph through NCX1-like would lead to a greater influx of extracellular Na+, the increased expression of the basolateral NKA was required to augment the capacity of intracellular Na+ homeostasis.
Asunto(s)
Bivalvos/fisiología , Calcificación Fisiológica , Calcio/química , Calcio/metabolismo , Células Epiteliales/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Biológico , Canales de Calcio/química , Hemolinfa , Homeostasis , Microscopía Fluorescente , Fotosíntesis , Filogenia , Isoformas de Proteínas , Agua de MarRESUMEN
The marble goby, Oxyeleotris marmorata, is a freshwater teleost, but can acclimate progressively to survive in seawater (salinity 30). As an obligatory air-breather, it can also survive long periods of emersion. Two isoforms of Na+/K+-ATPase (nka) α-subunit, nkaα1 and nkaα3, but not nkaα2, had been cloned from the gills of O. marmorata. The cDNA sequence of nkaα1 consisted of 3069 nucleotides, coding for 1023 amino acids (112.5 kDa), whereas nkaα3 consisted of 2976 nucleotides, coding for 992 amino acids (109.5 kDa). As only one form of branchial Nkaα1 was identified using molecular cloning in this study, O. marmorata lacks specific freshwater- and seawater-type Nkaα isoforms as demonstrated by some other euryhaline fish species. The nkaα1 transcript level was about 2.5-fold higher than that of nkaα3 in the gills of freshwater O. marmorata. During exposure to seawater, the branchial transcript level of nkaα1 increased significantly on day 1 (~3.3-fold) and day 6 (~2.6-fold). By contrast, the branchial transcript level of nkaα3 increased significantly on day 1 (~2.6-fold), but not on day 6, of seawater exposure. Six days of exposure to seawater also led to significant increases in protein abundances of Nkaα1 (~6.9-fold) and Nkaα3 (~2.8-fold) in the gills of O. marmorata. Hence, the mRNA and protein expressions of both nkaα1/Nkaα1 and nkaα3/Nkaα3 were up-regulated in O. marmorata during seawater acclimation. This could explain why Vmax increases but Km for Na+ and K+ remain unchanged in Nka extracted from the gills of O. marmorata acclimated to seawater as reported previously.
Asunto(s)
Aclimatación/fisiología , Branquias/enzimología , Isoenzimas/metabolismo , Perciformes/metabolismo , Agua de Mar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Isoenzimas/química , Isoenzimas/genética , Osmorregulación , Perciformes/clasificación , Perciformes/genética , Filogenia , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The fluted giant clam, Tridacna squamosa, can perform light-enhanced shell formation, aided by its symbiotic dinoflagellates (Symbiodinium, Cladocopium, Durusdinium), which are able to donate organic nutrients to the host. During light-enhanced shell formation, increased Ca2+ transport from the hemolymph through the shell-facing epithelium of the inner mantle to the extrapallial fluid, where calcification occurs, is necessary. Additionally, there must be increased absorption of exogenous Ca2+ from the surrounding seawater, across the epithelial cells of the ctenidium (gill) into the hemolymph, to supply sufficient Ca2+ for light-enhanced shell formation. When Ca2+ moves across these epithelial cells, the low intracellular Ca2+ concentration must be maintained. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) regulates the intracellular Ca2+ concentration by pumping Ca2+ into the sarcoplasmic/endoplasmic reticulum (SR/ER) and Golgi apparatus. Indeed, the ctenidium and inner mantle of T. squamosa, expressed a homolog of SERCA (SERCA-like transporter) that consists of 3009 bp, encoding 1002 amino acids of 110.6 kDa. SERCA-like-immunolabeling was non-uniform in the cytoplasm of epithelial cells of ctenidial filaments, and that of the shell-facing epithelial cells of the inner mantle. Importantly, the protein abundance of SERCA-like increased significantly in the ctenidium and the inner mantle of T. squamosa after 12 h and 6 h, respectively, of light exposure. This would increase the capacity of pumping Ca2+ into the endoplasmic reticulum and avert a possible surge in the cytosolic Ca2+ concentration in epithelial cells of the ctenidial filaments during light-enhanced Ca2+ absorption, and in cells of the shell-facing epithelium of the inner mantle during light-enhanced shell formation.
Asunto(s)
Exoesqueleto/metabolismo , Bivalvos/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Membrana/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Secuencia de Aminoácidos , Exoesqueleto/efectos de la radiación , Animales , Transporte Biológico/efectos de la radiación , Bivalvos/genética , Bivalvos/efectos de la radiación , Western Blotting , Regulación de la Expresión Génica/efectos de la radiación , Luz , Iluminación , Proteínas de Transporte de Membrana/genética , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Homología de Secuencia de AminoácidoRESUMEN
Nitrogen-deficient symbiotic dinoflagellates (zooxanthellae) living inside the fluted giant clam, Tridacna squamosa, need to obtain nitrogen from the host. Glutamine synthetase 1 (GS1) is a cytosolic enzyme that assimilates ammonia into glutamine. We determined the transcript levels of zooxanthellal GS1 (Zoox-GS1), which represented comprehensively GS1 transcripts of Symbiodinium, Cladocopium and Durusdinium, in five organs of T. squamosa. The outer mantle had significantly higher transcript level of Zoox-GS1 than the inner mantle, foot muscle, hepatopancreas and ctenidium, but the transcript ratios of Zoox-GS1 to zooxanthellal form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Zoox-rbcII), which represented the potential of ammonia assimilation relative to the phototrophic potential, were comparable among these five organs. Based on transcript ratios of Zoox-GS1 to zooxanthellal Urease (Zoox-URE), the outer mantle had the highest potential of urea degradation relative to ammonia assimilation among the five organs, probably because urea degradation could furnish CO2 and NH3 for photosynthesis and amino acid synthesis, respectively, in the symbionts therein. The protein abundance of Zoox-GS1 was upregulated in the outer mantle and the inner mantle during illumination. Zoox-GS1 could catalyze light-enhanced glutamine formation using ammonia absorbed from the host or ammonia released through urea degradation in the cytoplasm. The glutamine produced could be used to synthesize other nitrogenous compounds, including amino acids in the cytoplasm or in the plastid of the dinoflagellates. Some of the amino acids synthesized by the symbionts in the inner mantle and foot muscle could be donated to the host to support shell organic matrix formation and muscle production, respectively.
Asunto(s)
Amoníaco/metabolismo , Bivalvos/fisiología , Dinoflagelados/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Luz , Simbiosis , Aminoácidos/biosíntesis , Animales , Bivalvos/metabolismo , Especificidad de ÓrganosRESUMEN
The colorful outer mantle of giant clams contains abundance of symbiotic dinoflagellates (zooxanthellae) and iridocytes, and has direct exposure to light. In light, photosynthesizing dinoflagellates produce O2, and the host cells in the outer mantle would be confronted with hyperoxia-related oxidative stress. In comparison, the whitish inner mantle contains few symbiotic dinoflagellates and no iridocytes. It is involved in shell formation, and is shaded from light. CuZnSOD is a cytosolic enzyme that scavenges intracellular O2-. We had obtained from the outer mantle of the fluted giant clam, Tridacna squamosa, the complete cDNA coding sequence of a host-derived copper zinc superoxide dismutase (CuZnSOD), which comprised 462 bp and encoded for 154 amino acids with a calculated MW of 15.6 kDa. CuZnSOD was expressed strongly in the outer mantle, ctenidium, hepatopancreas and kidney. The transcript level of CuZnSOD remained unchanged in the outer mantle during light exposure, but the protein abundance of CuZnSOD increased ~3-fold after exposure to light for 6 or 12 h. By contrast, 12 h of light exposure had no significant effects on the gene and protein expression levels of CuZnSOD/CuZnSOD in the inner mantle. Hence, the increased expression of CuZnSOD in the outer mantle of T. squamosa was probably a host's response to ameliorate oxidative stress related to photosynthesis in the symbionts, and not simply due to increased metabolic rate in the host cells. Evidently, the host clam must possess light- or O2-responsive anti-oxidative defenses in order to align with the light-dependent photosynthetic activity of its symbionts.
Asunto(s)
Bivalvos/fisiología , Color , Luz , Proteínas/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Bivalvos/metabolismo , Bivalvos/efectos de la radiación , Dinoflagelados/metabolismo , Dinoflagelados/fisiología , FotosíntesisRESUMEN
Giant clams contain phototrophic zooxanthellae, and live in nutrient-deficient tropical waters where light is available. We obtained the complete cDNA coding sequence of a homolog of mammalian sodium/glucose cotransporter 1 (SGLT1) - SGLT1-like - from the ctenidium of the fluted giant clam, Tridacna squamosaSGLT1-like had a host origin and was expressed predominantly in the ctenidium. Molecular characterizations reveal that SGLT1-like of T. squamosa could transport urea, in addition to glucose, as other SGLT1s do. It has an apical localization in the epithelium of ctenidial filaments and water channels, and the apical anti-SGLT1-like immunofluorescence was stronger in individuals exposed to light than to darkness. Furthermore, the protein abundance of SGLT1-like increased significantly in the ctenidium of individuals exposed to light for 12â h, although the SGLT1-like transcript level remained unchanged. As expected, T. squamosa could perform light-enhanced glucose absorption, which was impeded by exogenous urea. These results denote the close relationships between light-enhanced glucose absorption and light-enhanced SGLT1-like expression in the ctenidium of T. squamosa Although glucose absorption could be trivial compared with the donation of photosynthates from zooxanthellae in symbiotic adults, SGLT1-like might be essential for the survival of aposymbiotic larvae, leading to its retention in the symbiotic stage. A priori, glucose uptake through SGLT1-like might be augmented by the surface microbiome through nutrient cycling, and the absorbed glucose could partially fulfill the metabolic needs of the ctenidial cells. Additionally, SGLT1-like could partake in urea absorption, as T. squamosa is known to conduct light-enhanced urea uptake to benefit the nitrogen-deficient zooxanthellae.
Asunto(s)
Bivalvos/metabolismo , Luz , Transportador 1 de Sodio-Glucosa/genética , Animales , Bivalvos/genética , Bivalvos/efectos de la radiación , Branquias/metabolismo , Glucosa/metabolismo , Análisis de Secuencia de ADN , Transportador 1 de Sodio-Glucosa/metabolismo , Urea/metabolismoRESUMEN
Giant clams live in nutrient-poor reef waters of the Indo-Pacific and rely on symbiotic dinoflagellates (Symbiodinium spp., also known as zooxanthellae) for nutrients. As the symbionts are nitrogen deficient, the host clam has to absorb exogenous nitrogen and supply it to them. This study aimed to demonstrate light-enhanced urea absorption in the fluted giant clam, Tridacna squamosa, and to clone and characterize the urea active transporter DUR3-like from its ctenidium (gill). The results indicate that T. squamosa absorbs exogenous urea, and the rate of urea uptake in the light was significantly higher than that in darkness. The DUR3-like coding sequence obtained from its ctenidium comprised 2346â bp, encoding a protein of 782 amino acids and 87.0â kDa. DUR3-like was expressed strongly in the ctenidium, outer mantle and kidney. Twelve hours of exposure to light had no significant effect on the transcript level of ctenidial DUR3-like However, between 3 and 12â h of light exposure, DUR3-like protein abundance increased progressively in the ctenidium, and became significantly greater than that in the control at 12â h. DUR3-like had an apical localization in the epithelia of the ctenidial filaments and tertiary water channels. Taken together, these results indicate that DUR3-like might participate in light-enhanced urea absorption in the ctenidium of T. squamosa When made available to the symbiotic zooxanthellae that are known to possess urease, the absorbed urea can be metabolized to NH3 and CO2 to support amino acid synthesis and photosynthesis, respectively, during insolation.
Asunto(s)
Bivalvos/metabolismo , Luz , Proteínas de Transporte de Membrana/metabolismo , Urea/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Bivalvos/genética , Dinoflagelados , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Transportadores de UreaRESUMEN
To obtain transcriptomic insights into branchial responses to salinity challenge in Anabas testudineus, this study employed RNA sequencing (RNA-Seq) to analyse the gill transcriptome of A. testudineus exposed to seawater (SW) for 6 days compared with the freshwater (FW) control group. A combined FW and SW gill transcriptome was de novo assembled from 169.9 million 101 bp paired-end reads. In silico validation employing 17 A. testudineus Sanger full-length coding sequences showed that 15/17 of them had greater than 80% of their sequences aligned to the de novo assembled contigs where 5/17 had their full-length (100%) aligned and 9/17 had greater than 90% of their sequences aligned. The combined FW and SW gill transcriptome was mapped to 13,780 unique human identifiers at E-value ≤1.0E-20 while 952 and 886 identifiers were determined as up and down-regulated by 1.5 fold, respectively, in the gills of A. testudineus in SW when compared with FW. These genes were found to be associated with at least 23 biological processes. A larger proportion of genes encoding enzymes and transporters associated with molecular transport, energy production, metabolisms were up-regulated, while a larger proportion of genes encoding transmembrane receptors, G-protein coupled receptors, kinases and transcription regulators associated with cell cycle, growth, development, signalling, morphology and gene expression were relatively lower in the gills of A. testudineus in SW when compared with FW. High correlation (R = 0.99) was observed between RNA-Seq data and real-time quantitative PCR validation for 13 selected genes. The transcriptomic sequence information will facilitate development of molecular resources and tools while the findings will provide insights for future studies into branchial iono-osmoregulation and related cellular processes in A. testudineus.
Asunto(s)
Branquias/metabolismo , Percas/metabolismo , Agua de Mar , Transcriptoma , Equilibrio Hidroelectrolítico , Animales , Simulación por Computador , Agua Dulce , Regulación de la Expresión Génica , Humanos , Osmorregulación , Reacción en Cadena en Tiempo Real de la Polimerasa , Salinidad , Análisis de Secuencia de ARNRESUMEN
The freshwater climbing perch, Anabas testudineus, is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia, this study aimed to obtain the complete cDNA coding sequences of various rhgp isoforms from the gills of A. testudineus, and to determine their mRNA and protein expression levels during 6â days of exposure to 100â mmolâ l-1 NH4Cl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four rhgp (rhag, rhbg, rhcg1 and rhcg2) had been identified from the gills of A. testudineus They had conserved amino acid residues for NH4+ binding, NH4+ deprotonation, channel gating and lining of the vestibules. Despite inwardly directed NH3 and NH4+ gradients, there were significant increases in the mRNA expression levels of the four branchial rhgp in A. testudineus at certain time points during 6â days of ammonia exposure, with significant increases in the protein abundances of Rhag and Rhcg2 on day 6. Immunofluorescence microscopy revealed a type of ammonia-inducible Na+/K+-ATPase α1c-immunoreactive ionocyte with apical Rhag and basolateral Rhcg2 in the gills of fish exposed to ammonia for 6â days. Hence, active ammonia excretion may involve NH4+ entering the ionocyte through the basolateral Rhcg2 and being excreted through the apical Rhag, driven by a transapical membrane electrical potential generated by the apical cystic fibrosis transmembrane conductance regulator Cl- channel, as suggested previously.
Asunto(s)
Amoníaco/metabolismo , Proteínas de Peces/genética , Glicoproteínas/genética , Perciformes/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Branquias/metabolismo , Branquias/fisiología , Glicoproteínas/química , Glicoproteínas/metabolismo , Perciformes/genética , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
Biosynthesis of ascorbate is known to occur in liver and/or kidney of some vertebrates; however, a recent study discovered the expression of l-gulono-γ-lactone oxidase, an enzyme essential for ascorbate synthesis, in the brain of the African lungfish, Protopterus annectens. This report provides an up-to-date review on ascorbate synthesis in fishes and the possible future directions of study in view of the discovery of the unusual site of ascorbate biosynthesis.
Asunto(s)
Ácido Ascórbico/biosíntesis , Peces/metabolismo , L-Gulonolactona Oxidasa/metabolismo , Animales , Ácido Ascórbico/metabolismo , Encéfalo/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Vertebrados/metabolismoRESUMEN
This study aimed to test the hypothesis that the brain of Protopterus annectens expressed L-gulono-γ-lactone oxidase (gulo/Gulo), the enzyme catalyzing the last step of ascorbate biosynthesis, and could maintain high concentrations of ascorbate during estivation. We cloned and sequenced gulo from the kidney of P. annectens and performed quantitative PCR to determine its mRNA expression in kidney and brain. Gulo activity was assayed and its protein abundance was determined by Western blot using custom-made anti-Gulo antibody. Effects of estivation on concentrations of ascorbate and dehydroascorbate in the kidney and brain were also determined. Both brain and kidney, but not liver, of P. annectens expressed gulo/Gulo. Desiccation induced P. annectens to estivate, and 6 mo of estivation led to drastic decreases in gulo/Gulo expression and ascorbate concentration in the kidney. However, high concentrations of ascorbate and ascorbate + dehydroascorbate were maintained in the brain during estivation, probably resulting from in situ ascorbate synthesis. Control fish were placed in freshwater, where they were fully active in a favorable environment unlike estivation on land. The ability to synthesize ascorbate to ameliorate oxidative stress directly in the brain might contribute to the ability of P. annectens to undergo prolonged estivation on land.
Asunto(s)
Ácido Ascórbico/biosíntesis , Encéfalo/enzimología , Estivación/fisiología , Peces/fisiología , Riñón/enzimología , L-Gulonolactona Oxidasa/biosíntesis , Secuencia de Aminoácidos , Animales , Agua Corporal , Secuencia Conservada , L-Gulonolactona Oxidasa/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Estrés Oxidativo , Filogenia , ARN Mensajero/biosíntesis , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
This study aimed to sequence and characterize two pro-coagulant genes, coagulation factor II (f2) and fibrinogen gamma chain (fgg), from the liver of the African lungfish Protopterus annectens, and to determine their hepatic mRNA expression levels during three phases of aestivation. The protein abundance of F2 and Fgg in the liver and plasma was determined by immunoblotting. The results indicated that F2 and Fgg of P. annectens were phylogenetically closer to those of amphibians than those of teleosts. Three days of aestivation resulted in an up-regulation in the hepatic fgg mRNA expression level, while 6â days of aestivation led to a significant increase (3-fold) in the protein abundance of Fgg in the plasma. Hence, there could be an increase in the blood-clotting ability in P. annectens during the induction phase of aestivation. By contrast, the blood-clotting ability in P. annectens might be reduced in response to decreased blood flow and increased possibility of thrombosis during the maintenance phase of aestivation, as 6â months of aestivation led to significant decreases in mRNA expression levels of f2 and fgg in the liver. There could also be a decrease in the export of F2 and Fgg from the liver to the plasma so as to avert thrombosis. Three to 6â days after arousal from 6â months of aestivation, the protein abundance of F2 and Fgg recovered partially in the plasma of P. annectens; a complete recovery of the transcription and translation of f2/F2 in the liver might occur only after refeeding.
Asunto(s)
Estivación/genética , Fibrinógeno/genética , Peces/genética , Regulación de la Expresión Génica , Hígado/metabolismo , Protrombina/genética , ARN Mensajero/genética , Aire , Animales , Fibrinógeno/metabolismo , Peces/metabolismo , Agua Dulce , Filogenia , Protrombina/metabolismo , ARN Mensajero/metabolismoRESUMEN
We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.
Asunto(s)
Estructuras Animales/citología , Peces/anatomía & histología , Granulocitos/citología , Linfocitos/citología , Estructuras Animales/ultraestructura , Animales , Estivación/fisiología , Peces/fisiología , Agua Dulce , Granulocitos/ultraestructura , Linfocitos/ultraestructuraRESUMEN
The objective of this study was to examine the effects of 6 days of emersion on nitrogen metabolism and excretion in the Chinese soft-shelled turtle, Pelodiscus sinensis. Despite having a soft shell with a cutaneous surface that is known to be water permeable, P. sinensis lost only ~2% of body mass and was able to maintain its hematocrit and plasma osmolality, [Na(+)] and [Cl(-)] during 6 days of emersion. During emersion, it ameliorated water loss by reducing urine output, which led to a reduction (by 29-76%) in ammonia excretion. In comparison, there was a more prominent reduction (by 82-99%) in urea excretion during emersion due to a lack of water to flush the buccopharyngeal epithelium, which is known to be the major route of urea excretion. Consequently, emersion resulted in an apparent shift from ureotely to ammonotely in P. sinensis. Although urea concentration increased in several tissues, the excess urea accumulated could only account for 13-22% of the deficit in urea excretion. Hence, it can be concluded that a decrease (~80%) in urea synthesis occurred in P. sinensis during the 6 days of emersion. Indeed, emersion led to significant decreases in the activity of some ornithine-urea cycle enzymes (argininosuccinate synthetase/argininosuccinate lyase and arginase) from the liver of P. sinensis. As a decrease in urea synthesis occurred without the accumulation of ammonia and total free amino acids, it can be deduced that ammonia production through amino acid catabolism was suppressed with a proportional reduction in proteolysis in P. sinensis during emersion. Indeed, calculated results revealed that there could be a prominent decrease (~88%) in ammonia production in turtles after 6 days of emersion. In summary, despite being ureogenic and ureotelic in water, P. sinensis adopted a reduction in ammonia production, instead of increased urea synthesis, as the major strategy to ameliorate ammonia toxicity and problems associated with dehydration during terrestrial exposure.
Asunto(s)
Amoníaco/metabolismo , Exoesqueleto/anatomía & histología , Inmersión , Nitrógeno/metabolismo , Tortugas/anatomía & histología , Tortugas/metabolismo , Urea/metabolismo , Aminoácidos/metabolismo , Amoníaco/orina , Animales , Arginasa/metabolismo , Argininosuccinatoliasa/metabolismo , Argininosuccinato Sintasa/metabolismo , Peso Corporal , China , Cloruros/sangre , Glutamato Deshidrogenasa/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Hematócrito , Riñón/metabolismo , Hígado/enzimología , Concentración Osmolar , Sodio/sangre , Tortugas/sangreRESUMEN
We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6 months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.
Asunto(s)
Estivación/fisiología , Peces/anatomía & histología , Peces/fisiología , Agua Dulce , Bazo/citología , Bazo/ultraestructura , África , Animales , Femenino , MasculinoRESUMEN
Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.
Asunto(s)
Aclimatación/fisiología , Adaptación Fisiológica/fisiología , Amoníaco/metabolismo , Agua Dulce , Percas/fisiología , Agua de Mar , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Branquias/metabolismo , Homeostasis/fisiología , Isoenzimas/fisiología , Subunidades de Proteína/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
The Chinese soft-shelled turtle, Pelodiscus sinensis, is well adapted to aquatic environments, including brackish swamps and marshes. It is ureotelic, and occasionally submerges its head into puddles of water during emersion, presumably for buccopharyngeal respiration. This study was undertaken to test the hypothesis that the buccophyaryngeal cavity constitutes an important excretory route for urea in P. sinensis. Results indicate that a major portion of urea was excreted through the mouth instead of the kidney during immersion. When restrained on land, P. sinensis occasionally submerged their head into water (20-100 min), during which urea excretion and oxygen extraction occurred simultaneously. These results indicate for the first time that buccopharyngeal villiform processes (BVP) and rhythmic pharyngeal movements were involved in urea excretion in P. sinensis. Urea excretion through the mouth was sensitive to phloretin inhibition, indicating the involvement of urea transporters (UTs). In addition, saliva samples collected from the buccopharyngeal surfaces of P. sinensis injected intraperitoneally with saline contained ~36 mmol N l(-1) urea, significantly higher than that (~2.4 mmol N l(-1)) in the plasma. After intraperitoneal injection with 20 µmol urea g(-1) turtle, the concentration of urea in the saliva collected from the BVP increased to an extraordinarily high level of ~614 µmol N ml(-1), but the urea concentration (~45 µmol N ml(-1)) in the plasma was much lower, indicating that the buccopharyngeal epithelium of P. sinensis was capable of active urea transport. Subsequently, we obtained from the buccopharyngeal epithelium of P. sinensis the full cDNA sequence of a putative UT, whose deduced amino acid sequence had ~70% similarity with human and mouse UT-A2. This UT was not expressed in the kidney, corroborating the proposition that the kidney had only a minor role in urea excretion in P. sinensis. As UT-A2 is known to be a facilitative urea transporter, it is logical to deduce that it was localized in the basolateral membrane of the buccopharyngeal epithelium, and that another type of primary or secondary active urea transporter yet to be identified was present in the apical membrane. The ability to excrete urea through the mouth instead of the kidney might have facilitated the ability of P. sinensis and other soft-shelled turtles to successfully invade the brackish and/or marine environment.