RESUMEN
Individuals of colonial animals (e.g. zooids) are in continuous turnover. In ascidians colonial or solitary species have evolved by convergence multiple times. Colonial Botryllus and Botrylloides are well-studied genera that exhibit colony-wide developmental mechanisms that regulate synchronous and orchestrated cycles of budding and turnover of zooids. The origins of modular developmental mechanisms that facilitated the evolution of coloniality in this group remain unclear. To reconstruct ancestral states of coloniality we studied Symplegma brakenhielmi, a sister taxon of the botryllids. S. brakenhielmi zooids are embedded in a common tunic and present a similar vascular system as the botrylloides, however development and turnover of zooids occurs asynchronously and in a more independent manner. We generated a table of common stages of budding in Symplegma and Botryllus for comparative studies of asexual development. We tested dependent processes of budding among individuals of the colony by systemic bud or zooid removals. Although our results showed a higher degree of independence in bud development in S. brakenhielmi, we found a subtle colony-wide regulatory mechanism of modular development, i.e. new buds expedited development after the removal of all buds in the colony. Next, we characterized external morphology, ultrastructure, and abundance of circulatory blood cells in the vascular system of S. brakenhielmi. Macrophage-like cells (MLCs) are involved in zooid resorption and turnover. Proportions of MLCs in the blood of S. brakenhielmi corresponded to the peak of occurrence of this cell type during the budding cycle of B. schlosseri. We found several new blood cell types in S. brakenhielmi, including two cell types that resemble circulatory progenitor stem cells of other botryllid colonial ascidians. These cells showed features of undifferentiated cells and expressed mitotic marker Phospho-histone H3. Comparative studies of S. brakenhielmi and B. schlosseri allow us to discuss possible changes in the regulation of modular development (i.e. regulation of life and death in the colony), and a possible contribution of circulatory blood cells in budding processes. We propose that the higher degree of developmental independence in S. brakenhielmi budding is a result of its ancestral solitary mode of development.
Asunto(s)
Evolución Biológica , Embrión no Mamífero/fisiología , Urocordados/embriología , Animales , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/ultraestructura , Hemocitos/citología , Hemocitos/ultraestructura , Estadios del Ciclo de Vida , Urocordados/citología , Urocordados/ultraestructuraRESUMEN
Nitric oxide (NO) production in ascidians is related to immune responses of blood cells, and also to events such as egg fertilization and notochord regression. However, the signaling pathway for NO generation has been little investigated in this animal model. The present contribution identifies the cells involved in NO production and provides new information about a pathway for NO signaling. We were able to identify eight types of blood cells in the hemolymph of the ascidian Phallusia nigra, of which signet ring cells, univacuolar refractile granulocytes, and morula cells were involved in NO production. Zymosan A and lipopolysaccharide (LPS) enhanced NO production by blood cells, and the compound N-nitro-L-arginine-methyl ester (L-NAME) reduced NO production. Finally, the application of protein kinase A (PKA) and protein kinase C (PKC) inhibitors revealed that these molecules participate, together with NFκB, in the regulation of NO production by blood cells of P. nigra. This is the first report to show that PKA and PKC are involved in a signaling pathway that leads to NO production in ascidian blood cells.
Asunto(s)
Hemocitos/metabolismo , Óxido Nítrico/biosíntesis , Urocordados/citología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Hemocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , NG-Nitroarginina Metil Éster/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Zimosan/farmacologíaRESUMEN
Norepinephrine (NE) is a neuro-hormone released by vertebrates and invertebrates during acute stress, and can influence their immune function. We found that NE depressed the production of nitric oxide (NO) by the hemocytes of ascidians. Our results with a fluorescent indicator for NO in assays using both NE and either α or ß-antagonist revealed that NE down-regulated NO production by the ascidian hemocytes. Our data suggest that NE may be acting via specific hemocyte receptors to induce a decrease in immune function.
Asunto(s)
Hemocitos/efectos de los fármacos , Óxido Nítrico/biosíntesis , Norepinefrina/farmacología , Urocordados/efectos de los fármacos , Antagonistas Adrenérgicos alfa/farmacología , Animales , Hemocitos/inmunología , Hemocitos/metabolismo , Norepinefrina/antagonistas & inhibidores , Fentolamina/farmacología , Urocordados/citología , Urocordados/inmunología , Urocordados/metabolismoRESUMEN
Ascidian hemolymph contains various types of blood cells (hemocytes), which are believed to be involved in defense mechanisms. We have studied nitric-oxide (NO) synthase activity in hemocytes of the ascidian Styela plicata after exposure to lipopolysaccharide (LPS). To investigate which cell types are involved in NO production, we first identified, by electron microscopy, the types of hemocytes previously described, mainly by light microscopy, by others. Five types of blood cells could be recognized in the hemolymph: granulocytes, hemoblasts, lymphocyte-like cells, morula cells, and pigment cells. The lymphocyte-like cells produced the most NO. In agreement with studies of other invertebrates, nitrite generation did not change after LPS stimulation in assays in vitro, under either different concentrations of LPS or different time periods. Therefore, we performed an in vivo assay by injecting a known quantity of Escherichia coli into the tunic of the ascidians in order to investigate possible differences in NO levels. No increase of NO occurred accompanying the inflammatory reaction suggesting that another molecule in the pathway was involved. We found that nuclear factor kappaB (NFkappaB) was activated. Since NFkappaB is involved in the production of many substances related to immune responses, additional molecules might also be generated in response to E. coli infection. These observations may improve our understanding of the reaction of animals to eutrophic conditions.
Asunto(s)
Hemocitos/metabolismo , Óxido Nítrico/biosíntesis , Urocordados , Animales , Activación Enzimática , Hemocitos/efectos de los fármacos , Hemocitos/ultraestructura , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Urocordados/citología , Urocordados/metabolismoRESUMEN
In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing approximately 300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 +/- 13.5% (mean +/- SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 +/- 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 +/- 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Antitrombinas/aislamiento & purificación , Heparina/aislamiento & purificación , Oocitos/química , Urocordados/química , Trombosis de la Vena/prevención & control , Animales , Anticoagulantes/uso terapéutico , Antitrombinas/uso terapéutico , Modelos Animales de Enfermedad , Inhibidores del Factor Xa , Femenino , Hemorragia/tratamiento farmacológico , Heparina/uso terapéutico , Masculino , Tiempo de Tromboplastina Parcial , Ratas , Ratas Wistar , Porcinos , Urocordados/citologíaRESUMEN
In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10 percent of mammalian heparin and about 5 percent as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5 percent (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4 percent, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13 percent without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.
Asunto(s)
Animales , Masculino , Femenino , Ratas , Anticoagulantes/aislamiento & purificación , Antitrombinas/aislamiento & purificación , Heparina/aislamiento & purificación , Oocitos/química , Urocordados/química , Trombosis de la Vena/prevención & control , Anticoagulantes/uso terapéutico , Antitrombinas/uso terapéutico , Modelos Animales de Enfermedad , Factor Xa/antagonistas & inhibidores , Hemorragia/tratamiento farmacológico , Heparina/uso terapéutico , Tiempo de Tromboplastina Parcial , Ratas Wistar , Porcinos , Urocordados/citologíaRESUMEN
In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.