The South American gymnotiform knifefish, Brachyhypopomus gauderio is a weakly electric fish species possessing an electric organ (EO). Development of the EO at embryonic and early larval stages has not been extensively studied. B. gauderio is relatively easy to keep in the aquarium and to obtain embryos and therefore could be a good animal model for studying development of the EO. However, little is known about its embryonic development. In this study, we described fertilisation in B. gauderio until early larval stage including 32 stages (180hpf), and determined the larval hatching that was between 3-4 days. The analyses showed a large EO at the ventral side of the muscle (veo). We also discovered another small potential EO-like tissue located at the dorsal side of the muscle (deo) and that has not been previously described. The results also described the derivative electrocytes from the electroprimordium which are separated from the ventral somite muscle in the 3.5dpf during the hatching period. This structure is further separated in the following days. At that time, the first myogenic EO is developed at the border between the ventral somite and the ventral tail fin. During progression of electroprimordium development, the location of the electroprimordium moves down ventrally in the tail fin where newly formed electrocytes appear with multiple nuclei seen in a large syncytial cell. Fluorescent immunohistochemistry showed that MF20 (myosin heavy chain) antibody stained muscles at stages 60hpf, 84hpf, 5.5dpf and 7.5dpf. MF20 also weakly stained the border of electrocytes and the electroprimordium at stages 5.5dpf and 7.5dpf. PAX7 antibody was expressed in the electroprimordium at stages 5.5dpf and 7.5dpf. Moreover, in situ hybridisation staining with scn4aa suggests that cell fate specification of EO may start even earlier at embryonic stage (40hpf). We investigated signalling pathways in EO development using specific inhibitors or activator for key signalling pathways (Bmp, Nodal, Fgf, Retionic Acid and Sonic hedgehog) to the B. gauderio at early stages using in situ hybridisation with scn4aa probe. The results show that Bmp inhibitor, FGF inhibitor and RA suppressed or reduced the scn4aa expression but also reduced the tail and tail fin development. Therefore it was not obvious if these signalling pathways are directly involved in the EO development or if the signals indirectly regulate the EO development by altering cell fates of tail tissues. The SHH inhibitor, cyclopamine treatment at late blastula stage suppressed scn4aa. These results suggest that sonic hedgehog at the late blastula to early gastrula stage has a crucial role in the EO development. Edar morpholino injection suppressed the dorsal EO via deletion of ventral and dorsal fins which was confirmed by the suppression of scn4aa expression in the morphant. This could be a result of the absence of the fin which may possibly be important for activating scn4aa in the dorsal side. To study EO development in the adult fish, the caudal filament was amputated. We observed that tail regeneration occurred during the following two weeks including newly regenerated EO, suggesting that this system would be useful for studying EO development using adult fish, and for learning mechanisms of tissue regeneration in the fish. In the current study, Zn-12 antibody detected a neurogenic EO of electric fish B. gauderio that has not been discovered before. The staining signal with Zn-12 was obvious in the neurogenic EO at adult stage, however the signal at 25dpf was faint and small, and was not detected in the larvae suggesting this organ is specific to juvenile to adult stage. Furthermore, detailed description of the EO structure at early and late stages were conducted using TEM analysis, showing nuclei, ribosomes, glycogen, endoplasmic reticulum, late endosomes and large number of mitochondrion from embryonic electroprimordium to adult EO. At 8.5dpf, the electroprimordium was seen in the front line of the EO moving towards the ventral end of the fin during the EO development. TEM analyses also showed intense myelin layers formed in the neourocytes of neurogenic EO at adult stage (7mpf) considering its important in neourocytes structure. Throughout these studies, we found that the knifefish, B. gauderio embryos and adult fish with the regeneration assay are excellent models for studying development of the electric organ.

Eigenmannia virescens is a gymnotiform weakly electric fish which uses a quasi-sinusoidal electric organ discharge (EOD) to sense their environment. EOD frequency (EODF) is individual-specific. In conspecific interactions, each fish perceives the EODF of the conspecific as a periodic amplitude modulation (AM) of their own discharge. When both fish are stationary, the depth of this AM is constant, but it varies when fish are swimming. We hypothesized that AM variations during swimming act as a noise source that could have no effect on, hinder, or enhance EODF identification. To test this, we quantified the jamming avoidance response (JAR) (a natural behaviour wherein fish are required to accurately determine one another’s EODF) in response to stimuli of varying depths of noise. These experiments demonstrated that swimming noise does not impair the ability of E. virescens to identify conspecific EODF, and actually improves its ability to detect the presence of a neighbouring fish.

The weakly electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. The EOD pulse is mainly shaped by sodium currents. There are two sodium channel α subunits orthologs of the mammalian Nav1.4 expressed in the EO of Sternopygus. Previous studies identified a novel splice variant of the Nav1.4b (Nav1.4bL), in which an extra 51-amino acid occurs in the N terminal end. Nav1.4bL currents inactivate and recover from inactivation significantly faster than Nav1.4bS. The voltage-dependence of steady-state inactivation of smNav1.4bL shifts to hyperpolarized potential. Structural analysis predicts an α-helix in the middle of the extended N terminus. Removal of a proline right after the α-helix significantly slows down current decay but has no effect on channel recovery from inactivation, suggesting inactivation and recovery have independent mechanism. Mutagenesis analysis of the extended N terminus showed that the short helical region, especially the positive charges in the helix, is an important determinant for channel voltage-dependence of steady-state inactivation. However, other residues outside the helical region are required for regulation of fast inactivation and recovery form inactivation. Functional and structural analysis provides evidence for the importance of the C terminus in fish Nav1.4b channel properties. Chimera in which the C terminus of smNav1.4bS was substituted by the human Nav1.4 C terminus, shows an 11 mV positive shift in voltage-dependence of activation and a -16 mV negative shift in inactivation. Deletion of the distal half of smNav1.4bS negatively shifted voltage-dependence of inactivation and significantly accelerated channel recovery from inactivation. In the deletion mutant, the regulation by the N segment is missing. Substitution of the C terminus mutant retains wild type channel inactivation and recovery properties and can be regulated by N segment again. My study provides evidence that the extended N terminus of smNav1.4bL binds the distal part of C terminal tail to modulate channel inactivation properties. This is the first time to show the distal C terminus is involved in channel recovery from inactivation. Studies in the fish sodium channel properties provide useful information to understand function and structure of voltage-gated sodium channels. / text

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [104]-122).

In 1800, Volta, inspired by the electric organs of a genus of electric fish, the Electrophorus, invented the first electric batteries, which were termed "artificial electric organs." Since then, the far-reaching implications of the fishes’ electrogenesis have come under greater attention and interest. In these fishes, the electric organ resembles a series of batteries. The electric organs are formed by electrocytes (the "batteries") with a distinct cytomorphology for discharging and charging. Although the arrangements of electrocytes in the electric organ are well-understood, the mechanisms involved in generating electric discharges within equivalent circuits remain unclear. In this thesis, the first element consists of adapting spatially defined models that we use to investigate the process of electrocyte charging and recharging under the added assumption of ion homeostasis, the process by which a cell restores its internal milieu. The study focuses on Eigenmannia and Electrophorus, two genera of electric fish. Eigenmannia's steady high-frequency dipole oscillator-like electric organ discharges enables electro-sensing and electro-communication, whilst Electrophorus's brief taser-like electric bursts serve as tetanizing predatory assaults. In addition, the second section of this study proposes a one-dimensional charge difference model that focuses on the modification of endogenous electric fields resulting from the uneven distribution of ions in a homeostatic apparatus.

Les trois projets que j’ai développés au cours de ma thèse s’inscrivent dans un même contexte, celui de l’étude de l’acétylcholinestérase (AChE) et des molécules apparentées à l’AChE au plan structural ou fonctionnel. Existence, identification et caractérisation du fonctionnement d’une « porte arrière » dans l’AChE. Caractérisation de la « protéine X », un récepteur non AChE de la fasciculine. Recherche d’un partenaire protéique endogène de l’AChE dans le cerveau de rat. / The three projects I developed during my thesis are related to the study of acetylcholinesterase (AChE) and of molecules related to AChE in their function or structure. Existence, identification and characterization of the functioning of a back door in AChE. Characterization of “protein X”, a non-AChE receptor for fasciculin. Search for an endogenous proteic partner of AChE in rat brain.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

In dieser Arbeit wurden die Morpho-Physiologie und das Verhalten zweier Arten Afrikanischer schwach elektrischer Fische, Marcusenius victoriae und Petrocephalus degeni, im Labor und in einem ihrer natürlichen Habitate im Lwamunda Sumpf in Uganda untersucht. Die zwei Hauptziele dieser Arbeit waren (i) tageszeitabhängige Verhaltensrhythmen (Aktivität, Habitatnutzung) im Labor und im Freiland zu untersuchen und (ii) die Ausprägung und Plastizität der morpho-physiologischen Merkmale von P. degeni zu untersuchen, die ihnen erlauben bei natürlich vorkommender, geringer Sauerstoffverfügbarkeit (Hypoxie) zu überleben. Tageszeitabhängige Verhaltensrhythmen beider Arten wurden im Labor über 42 Stunden und im natürlichen Habitat dieser Fische für sechs Tage erfasst. In den Laborversuchen verbrachten beide Arten tagsüber annähernd 100% der Zeit in einem bereitgestellten Versteck und schwammen nachts heraus um aktiv ihre Umwelt zu erkunden. Im Habitat wurden die meisten Fische in strukturell komplexen Habitaten unter schwimmenden Pflanzen detektiert. Nachts schwammen die Fische aktiv in die offenen und ungeschützten Bereiche der Lagune, vermutlich um nach Futter zu suchen und zu interagieren. Die Begleitende in-situ Messung der Sauerstoffverfügbarkeit zeigte, dass beide Arten präsent und vermutlich sogar am aktivsten waren während Phasen extremer nächtlicher Hypoxie. Zur Untersuchung der respiratorischen Merkmale von P. degeni wurden Respirometrieversuche mit hypoxie-akklimatisierten Tieren durchgeführt, Hämoglobin- und Laktatkonzentration im Blut gemessen, und morphologische Parameter an den ersten beiden Kiemenbögen erfasst. Die Fische zeigten niedrige Sauerstoffverbrauchsraten, welche sie bis zu einem sehr niedrigem äußeren Sauerstoffpartialdruck aufrechterhielten. Zusätzlich zeigten sie hohe Hämoglobin- und Laktatkonzentrationen im Blut. Bis zu 75 Tage Normoxie-Akklimatisierung führte zu reduzierter Hämoglobinkonzentration und kürzeren Kiemenfilamenten. / In this thesis, I investigated the morpho-physiology and behavior of two species of African mormyrid weakly electric fish, Marcusenius victoriae and Petrocephalus degeni, in the laboratory and in one of their natural habitats, the Lwamunda Swamp in Uganda. The two main objectives of this work were to (i) observe behavioral rhythms and habitat use patterns of both species under natural and laboratory conditions, and (ii) assess expression and plasticity of morpho-physiological traits that might enable P. degeni to survive naturally occurring low oxygen conditions (hypoxia). Behavioral rhythms were recorded in the laboratory over 42 hours and in the habitat on six sampling days. In the laboratory, both species spent close to 100% of the time in their shelter during the day and actively explore their environment at night. In the swamp lagoon, fish were most often encountered in structurally complex habitats under floating vegetation and ventured into open and unsheltered areas of the lagoon at night, presumably to forage and interact. Concomitant in-situ oxygen measurements revealed that these fish were present, and presumably most active during periods of extreme nocturnal hypoxia in their swamp habitat. To investigate respiratory traits of swamp-dwelling P. degeni, I conducted respirometry experiments and measured blood lactate and hemoglobin and gill morphometrics on the first two gill arches. Fish showed low routine oxygen consumption rates, which they maintained until a very low ambient oxygen partial pressure was reached. Additionally, they had high concentrations of hemoglobin and lactate in their blood. Up to 75 days of normoxia exposure reduced blood hemoglobin and gill filament length.