Electrophorus electricus as a model system for the study of membrane excitability

The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.     

Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus.     

Eel electric organ: hyperexpressing calmodulin system.

The electroplax of the electric eel Electrophorus electricus is the most abundant source of the calcium-binding protein calmodulin. The electroplax has 250 times the amount of calmodulin and its mRNA than eel skeletal muscle. Our data suggest that there is no major difference in gene copies, the degree of methylation, or genome rearrangement of the calmodulin gene in DNAs from eel electroplax and muscle. Differences in the calmodulin-binding proteins in electroplax and muscle suggest a differential role for the functional expression of calmodulin in cellular regulation.     

First draft genome sequence of the Japanese eel, Anguilla japonica

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species.     

Identification and distribution of chondroitin sulfate in the three electric organs of the electric eel, Electrophorus electricus (L.)

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Towards the diversification of lactococcal starter and non-starter species in mesophilic dairy culture systems

Lactococcus is one of the earliest identified fermentative bacterial genera and among its member species, the dairy-associated Lactococcus lactis and Lactococcus cremoris are undoubtedly the best studied. These two species are believed to have evolved from plant-associated lactococci and through genome decay and acquisition of plasmids, have adapted to the dairy niche. The past decade has witnessed a surge of activity in novel lactococcal species identification from insect, plant and animal sources. Currently, 22 Lactococcus species are described and in this review, we summarise the genome characteristics of and phylogenetic relationships among these species. Furthermore, we explore the role of mobile elements including plasmids and bacteriophages in the diversification of lactococcal species. The pace of identification of novel lactococcal species suggests that the number of lactococcal species is likely to continue to grow. With additional sequence data for the emerging species, it will be possible to perform pathogenicity/virulence risk evaluations and generate extensive insights into the niche adaptation strategies through which they have evolved.     

Theileria electrophorin.sp., a parasite of the electric eel Electrophorus electricus (Osteichthyes: Cypriniformes: Gymnotidae) from Amazonian Brazil

The name Theileria electrophori n.sp. is proposed for a small parasite described in the erythrocytes of the electric eel, Electrophorus electricus, from Amazonian Brazil. Division of the organism in the erythrocyte produces only four bacilliform daughter cells which become scattered in the host cell, without a cruciform or rosette-shaped disposition. Exoerythrocytic meronts producing a large number of merozoites were encountered in Giemsa-stained impression smears of the internal organs, principally in the liver, and are presumably the source of the intraerythrocytic forms of the parasite. This developmental pattern is characteristic of piroplasms within the family Theileriidae, where the author considers the parasite of E. electricus to most appropriately belong. It effectively distinguishes the organism from the dactylosomatid parasites Babesiosoma Jakowska and Nigrelli, 1956 and Dactylosoma Labbé, 1894 also found in fishes. This appears to be the second report of Theileria Bettencourt, Franca and Borges, 1907 in a fish.     

Acetylcholinesterases.

Acetylcholinesterase (E.C.3.1.1.7) is a widely distributed enzyme, particularly in excitable tissues such as muscle, nerve, and electric organs but also found in erythrocytes and snake venoms. The function of the enzyme at postsynaptic sites in excitable tissues is considered to be termination of synaptic transmission via the hydrolytic inactivation of acetylcholine. The functional role of the enzyme at extrajunctional sites of excitable tissues, in nerve endings and in the erythrocyte has not been established. In the past five or six years substantial progress has been made in terms of our understanding of acetylcholinesterases (AChE) particularly with regard to their molecular characterization, their subunit structure and their immunological properties. These advances have been due in part to successful purification of enzymes from various tissues by the application of affinity chromatography techniques. In addition, some progress has been made regarding physiological aspects of the development and regulation of AChE in excitable tissues. This review will focus on these aspects of AChE by reference to work utilizing the enzyme from the following sources: electric tissue of the eel, $\text{Electrophorus electricus}$, or electric fish, $\text{Torpedo}$, species; mammalian and avian skeletal muscle; neural tissues, particularly mammalian brain; and the mammalian erythrocyte. For more comprehensive reviews of AChE readers are referred to the following references (1, 2).     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

A review of changes in the fish assemblages of Levantine inland and marine ecosystems following the introduction of non-native fishes

The arrival of non‐native fishes in the Levant Basin began in the late 19th century. Whereas the presence of most of the 40 non‐native freshwater fishes stem from intentional introductions, either for aquaculture or pest control, the 62 species of non‐native marine fishes arrived by natural dispersal via the Suez Canal. Of the non‐native freshwater species, five have established successful breeding populations (mosquitofish Gambusia affinis, common carp Cyprinus carpio, crucian carp Carassius carassius, swordtail Xiphophorus hellerii and rainbow trout Oncorhynchus mykiss), and seven are regularly stocked in natural habitats (thinlip mullet Liza ramada, flathead mullet Mugil cephalus, European eel Anguilla anguilla, grass carp Ctenopharyngodon idella, Asian silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, black carp Mylopharyngodon piceus). Some non‐native species appear to have out‐competed native species. Gambusia affinis may have caused the extirpation of two native cyprinid fishes from the Qishon River basin (Levant silver carp Hemigrammocapoeta nana and common garra Garra rufa) and the southern Dead Sea (endemic Sodom's garra G. ghoerensis). The opening of the Suez Canal in 1869 allowed entry into the eastern Mediterranean of Indo‐Pacific and Erythrean biota, with the latter now dominating the community structure (50–90% of fish biomass) and function (altered native food web) of the Levantine littoral and infra‐littoral zones. The process has accelerated in recent years concurrent with a warming trend of the seawater. Record numbers of newly discovered non‐native species is leading to the creation of a human‐assisted Erythrean biotic province in the eastern Mediterranean.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Freshwater habitat use by a moray eel species,Gymnothorax polyuranodon,in Fiji shown by otolith microchemistry

Freshwater eels of the Anguillidae are diadromous because they migrate between ocean and freshwater environments, but other anguilliform fishes are generally considered to be strictly marine species. A few marine eels of the Muraenidae and Ophichthidae have occasionally been found in freshwater or estuaries, indicating that anguillids are not the only anguilliform eels that can use freshwater in some parts of the world. The moray eel Gymnothorax polyuranodon is one species that is known to be present in freshwater in the Indo-Pacific, but its life history is unknown. One way to evaluate what types of habitats are used by fishes is to determine the ratio of strontium (Sr) to calcium (Ca) in their otoliths, because this can show if they have used freshwater or saltwater environments. To evaluate the patterns of freshwater use by this unusual species of marine eel, the otolith Sr/Ca ratios of four G. polyuranodon (275–344 mm) caught in a freshwater stream of Fiji were analyzed. The consistently low Sr/Ca values (0–4) indicated upstream movement after settlement and freshwater or estuarine residence of all four individuals. These eels did not appear to have entered freshwater just for a short time period, which is consistent with other reports that this species is present in estuarine and freshwater habitats. This suggests that G. polyuranodon may be a catadromous species of marine eel. The similarities and differences between the life histories of anguillid eels and the few marine eels that have evolved the ability to invade freshwater habitats is discussed in relation to the evolutionary origin of diadromy in anguilliform fishes that originated in the marine environment.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

Implications of climate change for the fishes of the British Isles

Recent climatic change has been recorded across the globe. Although environmental change is a characteristic feature of life on Earth and has played a major role in the evolution and global distribution of biodiversity, predicted future rates of climatic change, especially in temperature, are such that they will exceed any that has occurred over recent geological time. Climate change is considered as a key threat to biodiversity and to the structure and function of ecosystems that may already be subject to significant anthropogenic stress. The current understanding of climate change and its likely consequences for the fishes of Britain and Ireland and the surrounding seas are reviewed through a series of case studies detailing the likely response of several marine, diadromous and freshwater fishes to climate change. Changes in climate, and in particular, temperature have and will continue to affect fish at all levels of biological organization: cellular, individual, population, species, community and ecosystem, influencing physiological and ecological processes in a number of direct, indirect and complex ways. The response of fishes and of other aquatic taxa will vary according to their tolerances and life stage and are complex and difficult to predict. Fishes may respond directly to climate‐change‐related shifts in environmental processes or indirectly to other influences, such as community‐level interactions with other taxa. However, the ability to adapt to the predicted changes in climate will vary between species and between habitats and there will be winners and losers. In marine habitats, recent changes in fish community structure will continue as fishes shift their distributions relative to their temperature preferences. This may lead to the loss of some economically important cold‐adapted species such as Gadus morhua and Clupea harengus from some areas around Britain and Ireland, and the establishment of some new, warm‐adapted species. Increased temperatures are likely to favour cool‐adapted (e.g. Perca fluviatilis) and warm‐adapted freshwater fishes (e.g. roach Rutilus rutilus and other cyprinids) whose distribution and reproductive success may currently be constrained by temperature rather than by cold‐adapted species (e.g. salmonids). Species that occur in Britain and Ireland that are at the edge of their distribution will be most affected, both negatively and positively. Populations of conservation importance (e.g.Salvelinus alpinus and Coregonus spp.) may decline irreversibly. However, changes in food‐web dynamics and physiological adaptation, for example because of climate change, may obscure or alter predicted responses. The residual inertia in climate systems is such that even a complete cessation in emissions would still leave fishes exposed to continued climate change for at least half a century. Hence, regardless of the success or failure of programmes aimed at curbing climate change, major changes in fish communities can be expected over the next 50 years with a concomitant need to adapt management strategies accordingly.     

The evolution of teleost pigmentation and the fish‐specific genome duplication

Teleost fishes have evolved a unique complexity and diversity of pigmentation and colour patterning that is unmatched among vertebrates. Teleost colouration is mediated by five different major types of neural‐crest derived pigment cells, while tetrapods have a smaller repertoire of such chromatophores. The genetic basis of teleost colouration has been mainly uncovered by the cloning of pigmentation genes in mutants of zebrafish Danio rerio and medaka Oryzias latipes. Many of these teleost pigmentation genes were already known as key players in mammalian pigmentation, suggesting partial conservation of the corresponding developmental programme among vertebrates. Strikingly, teleost fishes have additional copies of many pigmentation genes compared with tetrapods, mainly as a result of a whole‐genome duplication that occurred 320–350 million years ago at the base of the teleost lineage, the so‐called fish‐specific genome duplication. Furthermore, teleosts have retained several duplicated pigmentation genes from earlier rounds of genome duplication in the vertebrate lineage, which were lost in other vertebrate groups. It was hypothesized that divergent evolution of such duplicated genes may have played an important role in pigmentation diversity and complexity in teleost fishes, which therefore not only provide important insights into the evolution of the vertebrate pigmentary system but also allow us to study the significance of genome duplications for vertebrate biodiversity.     

Draft genome of the American Eel (Anguilla rostrata)

Freshwater eels (Anguilla sp.) have large economic, cultural, ecological and aesthetic importance worldwide, but they suffered more than 90% decline in global stocks over the past few decades. Proper genetic resources, such as sequenced, assembled and annotated genomes, are essential to help plan sustainable recoveries by identifying physiological, biochemical and genetic mechanisms that caused the declines or that may lead to recoveries. Here, we present the first sequenced genome of the American eel. This genome contained 305 043 contigs (N50 = 7397) and 79 209 scaffolds (N50 = 86 641) for a total size of 1.41 Gb, which is in the middle of the range of previous estimations for this species. In addition, protein‐coding regions, including introns and flanking regions, are very well represented in the genome, as 95.2% of the 458 core eukaryotic genes and 98.8% of the 248 ultra‐conserved subset were represented in the assembly and a total of 26 564 genes were annotated for future functional genomics studies. We performed a candidate gene analysis to compare three genes among all three freshwater eel species and, congruent with the phylogenetic relationships, Japanese eel (A. japanica) exhibited the most divergence. Overall, the sequenced genome presented in this study is a crucial addition to the presently available genetic tools to help guide future conservation efforts of freshwater eels.     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.