Growth and derived life-history characteristics of the Brazilian electric ray Narcine brasiliensis.

The majority of batoids are listed as Threatened (20.4%) or Data Deficient (41%) by the IUCN Red List. A key challenge to assessing Data-Deficient species is obtaining estimates of key life-history characteristics. Here, a Bayesian approach was used to estimate derived life-history characteristics from a growth model applied to the Data-Deficient Brazilian electric ray Narcine brasiliensis. The age of 170 specimens (107 females, 63 males) was estimated from vertebral centra, and total length, disc width, total weight and birth size were used in a joint estimation of sex-specific length-weight models and two-dimensional von Bertalanffy growth models. Estimates of age at length zero, age at maturity, longevity and mortality at age were derived simultaneously. The Bayesian joint modelling approach was robust to small sample sizes by adding a likelihood to constrain L0 and sharing parameters, such as Brody growth coefficient between length measurements. The median growth parameter estimates were a shared L0 = 38.8 mm, female L∞ = 515 mm, 𝑘 = 0.125 and male L∞ = 387 mm, 𝑘 = 0.194. Age at maturity was estimated to be 7.40-7.49 years for females and 4.45-4.47 years for males, whereas longevity was 22.5-22.6 years for females and 14.2 years for males depending on length measurement. Age-1 natural mortality was estimated to be 0.199-0.207 for females and 0.211-0.213 for males. The derived life-history characteristics indicate N. brasiliensis is earlier maturing, but slower growing relative to other Torpediniformes. These characteristics along with the species' endemism to southern Brazil and high by-catch rates indicate that one of the IUCN Red List threatened categories may be more appropriate for the currently Data-Deficient status. The Bayesian approach used for N. brasiliensis can prove useful for utilizing limited age-growth data in other Data-Deficient batoid species to inform necessary life characteristics for conservation and management.

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

BACKGROUND: The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. RESULTS: Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). CONCLUSIONS: In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.

Notes on the reproductive biology of the Brazilian electric ray Narcine brasiliensis (Elasmobranchii: Narcinidae).

This study provides information on the reproductive biology of Narcine brasiliensis based on 105 individuals (72 females and 33 males) sampled in São Paulo State, Brazil. The total length at maturity for females was 318·9 mm and for males was 279·8 mm; pregnant females were observed only during summer and autumn. The peak of the gonado-somatic index for females and condition factor for males in the spring suggest a preparation for pregnancy and a mating period during this season. The capture of immature individuals indicates a need for management of the species in this region.

Electroanaesthesia--from torpedo fish to TENS.

In 153 AD, the Roman physician Scribonius Largus identified that electric current had analgesic properties, instructing patients to stand on an electric ray for the treatment of gout. In 2014, transcranial magnetic stimulation was approved by the National Institute for Health and Care Excellence for the treatment of migraine. Although separated by nearly two millennia, these milestones represent the evolution of the utilisation of electric current in medical and anaesthetic practice. Significant advances have been made over the last century in particular, and during the 1960s and 1970s, tens of thousands of patients were reportedly anaesthetised for surgical interventions using electric current as the anaesthetic agent. Many medical interventions, including transcutaneous electrical nerve stimulation and deep brain stimulation, have evolved in the aftermath of investigations into electroanaesthesia; the potential for electric current to be an anaesthetic agent of the future still exists.

Comparative study on trace metal accumulation in the liver of two fish species (Torpedinidae): concentration-size relationship.

Metal concentrations (Hg, Cd, Pb, As, Cr, Cu, Zn and Ni) were measured in the liver of two fish, Torpedo nobiliana (electric ray) and Torpedo marmorata (marbled electric ray), from the Mediterranean Sea in order to comparatively investigate their current pollution status. Maximum mean levels of Hg were detected in electric ray (mean: 2.16µgg(-1) ww), while marbled electric ray accumulated especially Cd (mean: 0.06µgg(-1) ww), Cu (mean: 3.83µgg(-1) ww) and As (mean: 32.64µgg(-1) ww). The metal concentrations are similar to those reported in literature, except for Cd, As and Ni. Hg concentrations increased with increasing fish body length in both species, whilst no significant concentration-size relationship was found for other metals. To the best of our knowledge this is the first report providing information on trace metal levels and relationship between concentration and size of these cartilaginous fishes. Future studies on the concentrations and effects of environmental contaminants in various torpedinid species are surely needed.

Sink and swim: kinematic evidence for lifting-body mechanisms in negatively buoyant electric rays Narcine brasiliensis.

Unlike most batoid fishes, electric rays neither oscillate nor undulate their body disc to generate thrust. Instead they use body-caudal-fin (BCF) locomotion. In addition, these negatively buoyant rays perform unpowered glides as they sink in the water column. In combination, BCF swimming and unpowered gliding are opposite ends on a spectrum of swimming, and electric rays provide an appropriate study system for understanding how the performance of each mode is controlled hydrodynamically. We predicted that the dorso-ventrally flattened body disc generates lift during both BCF swimming and gliding. To test this prediction, we examined 10 neonate lesser electric rays, Narcine brasiliensis, as they swam and glided. From video, we tracked the motion of the body, disc, pelvic fins and tail. By correlating changes in the motions of those structures with swimming performance, we have kinematic evidence that supports the hypothesis that the body disc is generating lift. Most importantly, both the pitch of the body disc and the tail, along with undulatory frequency, interact to control horizontal swimming speed and Strouhal number during BCF swimming. During gliding, the pitch of the body disc and the tail also interact to control the speed on the glide path and the glide angle.

Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the ß2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/ß2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel ß subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.

Organization of the vitelline envelope in ovarian follicles of Torpedo marmorata Risso, 1810 (Elasmobranchii: Torpediniformes).

In Torpedo marmorata, the vitelline envelope (VE), an extracellular envelope surrounding the growing oocyte, consists of fibrils and amorphous materials that are deposited in the perivitelline space starting from the initial steps of oocyte growth. SDS-PAGE analysis of the isolated and purified VE reveals that it consists of different glycoproteins. Furthermore, our investigations showed that the 120 and 66 kDa glycoproteins are positive to an antibody directed against gp69/64 of the Xenopus laevis VE and are synthesized under the control of 17beta-estradiol in the liver, that, together follicle cells and the oocyte, is the biosynthetic site of VE components.

Steady-state coupling of ion-channel conformations to a transmembrane ion gradient.

Under stationary conditions, opening and closing of single Torpedo electroplax chloride channels show that the number of transitions per unit time between inactivated and conducting states are unequal in opposite directions. This asymmetry, which increases with transmembrane electrochemical gradient for the chloride ion, violates the principle of microscopic reversibility and thus demonstrates that the channel-gating process is not at thermodynamic equilibrium. The results imply that the channel's conformational states are coupled to the transmembrane electrochemical gradient of the chloride ion.

Tooth reorientation affects tooth function during prey processing and tooth ontogeny in the lesser electric ray, Narcine brasiliensis.

The dental anatomy of elasmobranch fishes (sharks, rays and relatives) creates a functional system that is more dynamic than that of mammalian dentition. Continuous dental replacement (where new teeth are moved rostrally to replace older ones) and indirect fibrous attachment of the dentition to the jaw allow teeth to reorient relative to the jaw over both long- and short-term scales, respectively. In this study, we examine the processing behavior and dental anatomy of the lesser electric ray Narcine brasiliensis (Olfers, 1831) to illustrate that the freedom of movement of elasmobranch dentition allows a functional flexibility that can be important for complex prey processing behaviors. From static manipulations of dissected jaws and observations of feeding events in live animals, we show that the teeth rotate during jaw protrusion, resulting in a secondary grasping mechanism that likely serves to hold prey while the buccal cavity is flushed free of sediment. The function of teeth is not always readily apparent from morphology; in addition to short-term reorientation, the long-term dental reorientation during replacement allows a given tooth to serve multiple functions during tooth ontogeny. Unlike teeth inside the mouth, the cusps of external teeth (on the portion of the tooth pad that extends past the occlusal plane) lay flat, such that the labial faces act as a functional battering surface, protecting the jaws during prey excavation.

Allosteric mechanisms in normal and pathological nicotinic acetylcholine receptors.

Recent chemical and advanced structural studies on site-directed and naturally occurring pathological mutants of individual members of the multigene family of nicotinic acetylcholine receptors have yielded structure-function relationships supporting indirect 'allosteric' interactions between the acetylcholine-binding sites and the ion channel in signal transduction.

ATP dependence of the non-specific ion channel in Torpedo synaptic vesicles.

Synaptic vesicles of Torpedo electromotor neurons contain a high amount of ATP. The concentration of total ATP is around 120 mM, whereas the free [ATP] is about 5-6 mM. We examined the effect of intravesicular ATP on the non-specific ion channel in Torpedo-fused synaptic vesicles. It was found that this channel is closed when the ATP concentration is above 2 mM, but it is very frequently open at lower ATP concentrations. Unmasking this ion channel at a low ATP concentration may be significant for post-fusion control of transmitter release by the 'kiss and run' mechanism in normal conditions, while during metabolic stress it may underlie dissipation of important gradients across the vesicle membrane.

Functional morphology of jaw trabeculation in the lesser electric ray Narcine brasiliensis, with comments on the evolution of structural support in the Batoidea.

The design of minimum-weight structures that retain their integrity under dynamic loading regimes has long challenged engineers. One solution to this problem found in both human and biological design is the optimization of weight and strength by hollowing a structure and replacing its inner core with supportive struts. In animals, this design is observed in sand dollar test, avian beak, and the cancellous bone of tetrapod limbs. Additionally, within the elasmobranch fishes, mineralized trabeculae (struts) have been reported in the jaws of durophagous myliobatid stingrays (Elasmobranchii: Batoidea), but were believed to be absent in basal members of the batoid clade. This study, however, presents an additional case of batoid trabeculation in the lesser electric ray, Narcine brasiliensis (Torpediniformes). The trabeculae in these species likely play different functional roles. Stingrays use their reinforced jaws to crush bivalves, yet N. brasiliensis feeds by ballistically protruding its jaws into the sediment to capture polychaetes. In N. brasiliensis, trabeculae are localized to areas likely to experience the highest load: the quadratomandibular jaw joints, hyomandibular-cranial joint, and the thinnest sections of the jaws immediately lateral to the symphyses. However, the supports perform different functions dependent on location. In regions where the jaws are loaded transversely (as in durophagous rays), "load leading" trabeculae distribute compressive forces from the cortex through the lumen of the jaws. In the parasymphyseal regions of the jaws, "truss" trabeculae form cross-braces perpendicular to the long axes of the jaws. At peak protrusion, the jaw arch is medially compressed and the jaw loaded axially such that these trabeculae are positioned to resist buckling associated with excavation forces. "Truss" trabeculae function to maintain the second moment of area in the thinnest regions of the jaws, illustrating a novel function for batoid trabeculation. Thus, this method of structural support appears to have arisen twice independently in batoids and performs strikingly different ecological functions associated with the distribution of extreme loading environments.

Loading and recycling of synaptic vesicles in the Torpedo electric organ and the vertebrate neuromuscular junction.

In vertebrate motor nerve terminals and in the electromotor nerve terminals of Torpedo there are two major pools of synaptic vesicles: readily releasable and reserve. The electromotor terminals differ in that the reserve vesicles are twice the diameter of the readily releasable vesicles. The vesicles contain high concentrations of ACh and ATP. Part of the ACh is brought into the vesicle by the vesicular ACh transporter, VAChT, which exchanges two protons for each ACh, but a fraction of the ACh seems to be accumulated by different, unexplored mechanisms. Most of the vesicles in the terminals do not exchange ACh or ATP with the axoplasm, although ACh and ATP are free in the vesicle interior. The VAChT is controlled by a multifaceted regulatory complex, which includes the proteoglycans that characterize the cholinergic vesicles. The drug (-)-vesamicol binds to a site on the complex and blocks ACh exchange. Only 10-20% of the vesicles are in the readily releasable pool, which therefore is turned over fairly rapidly by spontaneous quantal release. The turnover can be followed by the incorporation of false transmitters into the recycling vesicles, and by the rate of uptake of FM dyes, which have some selectivity for the two recycling pathways. The amount of ACh loaded into recycling vesicles in the readily releasable pool decreases during stimulation. The ACh content of the vesicles can be varied over eight-fold range without changing vesicle size.

An electric generator using living Torpedo electric organs controlled by fluid pressure-based alternative nervous systems.

Direct electric power generation using biological functions have become a research focus due to their low cost and cleanliness. Unlike major approaches using glucose fuels or microbial fuel cells (MFCs), we present a generation method with intrinsically high energy conversion efficiency and generation with arbitrary timing using living electric organs of Torpedo (electric rays) which are serially integrated electrocytes converting ATP into electric energy. We developed alternative nervous systems using fluid pressure to stimulate electrocytes by a neurotransmitter, acetylcholine (Ach), and demonstrated electric generation. Maximum voltage and current were 1.5 V and 0.64 mA, respectively, with a duration time of a few seconds. We also demonstrated energy accumulation in a capacitor. The current was far larger than that using general cells other than electrocytes (~pA level). The generation ability was confirmed against repetitive cycles and also after preservation for 1 day. This is the first step toward ATP-based energy harvesting devices.

The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.

Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 µM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 µM; fmoc-1, IC50, 10.00 ± 1.38 µM; fmoc-2, IC50, 16.67 ± 1.95 µM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.

Characteristics of the Na+/K+-ATPase from Torpedo californica expressed in Xenopus oocytes: a combination of tracer flux measurements with electrophysiological measurements.

The Na+/K+-ATPase from electroplax of Torpedo californica was incorporated into the plasma membrane of Xenopus oocytes by microinjection of mRNA coding for the alpha- and beta-subunit of the enzyme; the mRNAs were obtained by in vitro translation of cloned cDNAs (Noguchi et al. (1988) FEBS Lett. 225, 27-32). (1) Measurements of ouabain-sensitive membrane current revealed that the Na+/K+-ATPase of Torpedo is less sensitive to ouabain than the endogenous enzyme. (2) The ouabain-sensitive membrane currents in mRNA-injected oocytes exhibit similar voltage dependence as the currents generated by the endogenous ATPase of Xenopus oocytes; in particular, the current-voltage relation exhibits a maximum and a negative slope at potentials more positive than +20 mV. (3) A maximum can also be detected if the rate of 22Na+ efflux is determined under different voltage-clamp conditions. If membrane current and rate of Na+2 efflux are determined simultaneously, a voltage-independent ratio between current and flux is obtained suggesting voltage-independent Na+-K+ stoichiometry. The data are compatible with a 3Na+-2K+ stoichiometry.

Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB).

We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl(-)channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V = 60 mV) with an IC50 of approximately 30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl(-) for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.