The hearing abilities of the prawn Palaemon serratus

The mechanism of sound reception and the hearing abilities of the prawn (Palaemon serratus) have been studied using a combination of anatomical, electron microscopic and electrophysiological approaches, revealing that P. serratus is responsive to sounds ranging in frequency from 100 to 3000 Hz. It is the first time that the Auditory Brainstem Response (ABR) recording technique has been used on invertebrates, and the acquisition of hearing ability data from the present study adds valuable information to the inclusion of an entire sub-phylum of animals when assessing the potential impact of anthropogenic underwater sounds on marine organisms. Auditory evoked potentials were acquired from P. serratus, using two subcutaneous electrodes positioned in the carapace close to the supraesophageal ganglion and the statocyst (a small gravistatic organ located below the eyestalk on the peduncle of the bilateral antennules). The morphology of the statocyst receptors and the otic nerve pathways to the brain have also been studied, and reveal that P. serratus possesses an array of sensory hairs projecting from the floor of the statocyst into a mass of sand granules embedded in a gelatinous substance. It is the purpose of this work to show that the statocyst is responsive to sounds propagated through water from an air mounted transducer. The fundamental measure of the hearing ability of any organism possessing the appropriate receptor mechanism is its audiogram, which presents the lowest level of sound that the species can hear as a function of frequency. The statocyst of P. serratus is shown here to be sensitive to the motion of water particles displaced by low-frequency sounds ranging from 100 Hz up to 3000 Hz, with a hearing acuity similar to that of a generalist fish. Also, recorded neural waveforms were found to be similar in both amplitude and shape to those acquired from fish and higher vertebrates, when stimulated with low-frequency sound, and complete ablation of the electrophysiological response was achieved by removal of the statocyst.     

Voltage dependence of the [Ca2+](i) oscillations system, in the Mg2+ -stimulated oocyte of the prawn Palaemon serratus

By voltage-clamp technique and simultaneous [Ca2+](i)measurements, we studied the modifications, induced by changes in membrane voltage, in the pattern of the [Ca2+](i)oscillation period, displayed by the Mg2+-stimulated oocyte of the prawn Palaemon serratus. When the Mg2+-stimulated oocytes were voltage clamped at 0mV, they developed a [Ca2+](i)signal with a more pronounced oscillatory pattern than that obtained on unclamped oocytes. Indeed, they displayed a first peak followed by a series of sharp [Ca2+](i)transients and a prominent [Ca2+](i)oscillatory plateau. By contrast, oocytes voltage clamped at - 60mV showed a first peak followed by a stable high [Ca2+](i)level forming a long continuous plateau devoid of oscillations. By using caged InsP3, we established that the ER InsP3 receptor is not voltage sensitive. Paradoxically, we showed the voltage sensitivity of the Mg2+ receptor-signal transduction system which is more reactive to Mg2+ ions at -60mV than at 0mV. Using different calmodulin inhibitors of the PM CA pump such as trifluoperazin (100microM), W-7 (50microM) and calmidazolium (50microM), we suppressed the [Ca2+](i)oscillatory pattern in oocytes voltage clamped at 0mV. From these results we propose that this special voltage-dependent oscillatory system could be regulated by a significant involvement of the electrogenic PM CA pump.     

Extracellular Mg2+ induces a loss of microvilli, membrane retrieval, and the subsequent cortical reaction, in the oocyte of the prawn Palaemon serratus

Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

Ontogeny and optics of the eyes of the common prawn Palaemon (Palaemon) serratus (Pennant, 1777)

A brief review of the work on crustacean compound eyes is given. Two main types of eye have been recognized: apposition and superposition. The ontogeny of the eyes of the common prawn Palaemon serratus is examined using a variety of methods: photography of live specimens, histological sections, SEM and TEM. In common with other decapod larvae, the common prawn hatches with apposition eyes having circular lenses packed hexagonally. After metamorphosis the gradual squaring of the eye facets, begun during the larval phase, is completed. This is an essential prerequisite for the functioning of the facultative superposition reflecting optics found in long-bodied decapods (e.g. shrimps, prawns and lobsters) and some Anomura. The possible phylogenetical significance of superposition, reflecting optics is also discussed.     

Localization of serotonin-like immunoreactivity in the eyestalk of the prawn Palaemon serratus (Crustacea, Decapoda, Natantia)

A serotonin-like substance in the organ of Bellonci in the eyestalks of embryos, larvae, and adults of the prawn Palaemon serratus was visualized by the use of two specific antisera against serotonin (5-hydroxytryptamine; 5-HT) in combination with peroxidase-antiperoxidase (PAP). The organ of Bellonci, characterized by compact onion bodies distally and degenerating onion bodies proximally, was the only site of the serotonin-like substance in adults, as well as during development in embryos and larvae. Variations in the content of the 5-HT analogue in the adult were detected during the molting cycle. There was more immunoreactivity in specimens fixed at night than in those fixed in daytime. Likewise, colchicine and nialamide injections enhanced the immunoreactivity of the serotonin-like substance. Extirpations of the medulla externa X organ (MEX), a neurosecretory cell group of the optic ganglion medulla externa, produced the same effect.     

Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes

Intracellular Mg(2+) buffering and Mg(2+) extrusion were investigated in Xenopus laevis oocytes. Mg(2+) or EDTA were pressure injected and the resulting changes in the intracellular Mg(2+) concentration were measured simultaneously with Mg(2+)-selective microelectrodes. In the presence of extracellular Na(+), injected Mg(2+) was extruded from the oocytes with an estimated v(max) and K(M) of 74 pmol cm(-2)s(-1) and 1.28 mM, respectively. To investigate genuine cytosolic Mg(2+) buffering, measurements were carried out in the nominal absence of extracellular Na(+) to block Mg(2+) extrusion, and during the application of CCCP (inhibiting mitochondrial uptake). Under these conditions, Mg(2+) buffering calculated after both MgCl(2) and EDTA injections could be described by a buffer equivalent with a concentration of 9.8mM and an apparent dissociation constant, K(d-app), of 0.6mM together with an [ATP](i) of 0.9 mM with a K(d-app) 0.12 mM. Xenopus oocytes thus possess highly efficient mechanisms to maintain their intracellular Mg(2+) concentration.     

Immunocytochemical localization of a substance in the eyestalk of the prawn,Palaemon serratus,reactive with an anti-FMRF-amide rabbit serum

Summary By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.     

The CorA Mg2+ channel is required for the virulence of Salmonella enterica serovar typhimurium

CorA is the primary Mg²⁺ channel in Salmonella enterica serovar Typhimurium. A corA mutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in a corA mutant strain, which ultimately manifests itself as a decrease in virulence.     

Synaptic junctions in the sinus gland of the freshwater prawn Palaemon paucidens

Summary Two types of neurosecretory fibers, designated as Type 5 and Type 6 axons, in the sinus gland of the freshwater prawn, Palaemon, establish contact with other neurosecretory axons by means of synaptic junctions. This finding strongly supports the view that release of some neurohormones from the eyestalk may be regulated by neurosecretory neurons through synaptic transmission.The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study, to Mr. S. Kubota for collecting the material, and to Mr. Y. Takakuwa for his excellent assistance in electron microscopy     

Neurosecretory cell types in the eyestalk of the freshwater prawn Palaemon paucidens

Summary In the medulla terminalis ganglionic X-organ (MTGX) of the eyestalk of the freshwater prawn, Palaemon paucidens, six peptidergic neuro-secretory cell types (A-, B-, C-, D-, E-, and F-cells) are distinguishable on the basis of the different morphology of their elementary granules and rough endoplasmic reticulum (rER). All of these cell types seem to correspond to Type-IIIa cells or dispersing Type-IV cells, that have previously been differentiated at the light microscopic level (Hisano, 1974), as judged from the dimensions of their cell bodies and nuclei. Two other peptidergic neurosecretory cell types that are apparently comparable to the Type-II and Type-IIIb cells (Hisano, 1974), respectively, are recognized in parts of the optic ganglia other than MTGX, and these are now designated as Gand H-cells, respectively. All the remaining cell types, designated as Type-I, cluster-forming Type-IV, Type-V and Type-VI cells in our previous light microscopic study, have small cored-vesicles in their cytoplasm. It remains undecided whether these, possibly aminergic, neurons are neurosecretory or not.The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study.     

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.     

Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.     

Variations in the lactate dehydrogenase activity during the development of the shrimp Palaemon serratus

• 1.1. The lactate dehydrogenase (LDH) from Palaemon serratus muscle has been studied throughout the development of the animal.
• 2.2. Enzymatic activities have been traced by polyacrylamide gel electrophoresis and kinetic studies.
• 3.3. The existence of two enzymes (L₁ and L₂) has been demonstrated.
• 4.4. During the larval development, both L₁ and L₂ remain at a low level.
• 5.5. After the larvae hatch L₁ and L₂ gradually rise although L₁ is predominant.
• 6.6. Measurement of kinetic parameters shows that the general behaviour of the enzymes of the embryo resembles that of the adult enzymes.
• 7.7. However, one can observe during the development a constant increase in the affinity of the enzyme towards its substrate, lactate.

     

Immunocytochemical study of crustacean hyperglycemic hormone in the eyestalks of the prawn Palaemon serratus (Pennant) and some other Palaemonidae,in relation to variations in the blood glucose level

With the use of rabbit antisera against crustacean hyperglycemic hormone (CHH), it is possible to describe a distinct immunopositive reaction in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ₂ (MTGX₂), in the MTGX-sinus gland tract, and in a considerable part of the sinus gland from several species of prawns belonging to the Palaemonidae. By introductory studies on the CHH system in Palaemon serratus, we can postulate a sequence in the activity cycle of the CHH-producing cells on the basis of differences in staining intensity of the immunoreaction and such morphometric parameters as cellular and nuclear diameter. By studying the CHH-producing system in combination with variations in the glucose level of the blood, an “inverse relationship” is observed between the number of immunoreactive cells and the blood glucose level during different periods of the year as well as during different stages of the molting cycle. A “shift in phase” of this correlation during the diurnal cycle suggests that several rhythmical phenomena may play a role in the regulation of glycemia in Crustacea.     

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.     

Septin2 is modified by SUMOylation and required for chromosome congression in mouse oocytes

In mitosis, centrosomes nucleate microtubules that capture the sister kinetochores of each chromosome to facilitate chromosome congression. In contrast, during meiosis chromosome congression on the acentrosomal spindle is driven primarily by movement of chromosomes along laterally associated microtubule bundles. Previous studies have indicated that septin2 is required for chromosome congression and cytokinesis in mitosis, we therefore asked whether perturbation of septin2 would impair chromosome congression and cytokinesis in meiosis. We have investigated its expression, localization and function during mouse oocyte meiotic maturation. Septin2 was modified by SUMO-1 and its levels remained constant from GVBD to metaphase II stages. Septin2 was localized along the entire spindle at metaphase and at the midbody in cytokinesis. Disruption of septins function with an inhibitor and siRNA caused failure of the metaphase I /anaphase I transition and chromosome misalignment but inhibition of septins after the metaphase I stage did not affect cytokinesis. BubR1, a core component of the spindle checkpoint, was labeled on misaligned chromosomes and on chromosomes aligned at the metaphase plate in inhibitor-treated oocytes that were arrested in prometaphase I/metaphase I, suggesting activation of the spindle assembly checkpoint. Taken together, our results demonstrate that septin2 plays an important role in chromosome congression and meiotic cell cycle progression but not cytokinesis in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.