Gene and Allele-Specific Expression Underlying the Electric Signal Divergence in African Weakly Electric Fish.

In the African weakly electric fish genus Campylomormyrus, electric organ discharge signals are strikingly different in shape and duration among closely related species, contribute to prezygotic isolation, and may have triggered an adaptive radiation. We performed mRNA sequencing on electric organs and skeletal muscles (from which the electric organs derive) from 3 species with short (0.4 ms), medium (5 ms), and long (40 ms) electric organ discharges and 2 different cross-species hybrids. We identified 1,444 upregulated genes in electric organ shared by all 5 species/hybrid cohorts, rendering them candidate genes for electric organ-specific properties in Campylomormyrus. We further identified several candidate genes, including KCNJ2 and KLF5, and their upregulation may contribute to increased electric organ discharge duration. Hybrids between a short (Campylomormyrus compressirostris) and a long (Campylomormyrus rhynchophorus) discharging species exhibit electric organ discharges of intermediate duration and showed imbalanced expression of KCNJ2 alleles, pointing toward a cis-regulatory difference at this locus, relative to electric organ discharge duration. KLF5 is a transcription factor potentially balancing potassium channel gene expression, a crucial process for the formation of an electric organ discharge. Unraveling the genetic basis of the species-specific modulation of the electric organ discharge in Campylomormyrus is crucial for understanding the adaptive radiation of this emerging model taxon of ecological (perhaps even sympatric) speciation.

Diet in phenotypically divergent sympatric species of African weakly electric fish (genus: Campylomormyrus)-A hybrid capture/HTS metabarcoding approach.

Ecological speciation within the mormyrid genus Campylomormyrus resulted in sympatric species exhibiting divergence in their feeding apparatus and electric organ discharge (EOD). This study documents the overall diet of the genus Campylomormyrus and examines the hypothesis that the Campylomormyrus radiation is caused by adaptation to different food sources. We performed diet assessment of five sympatric Campylomormyrus species (C. alces, C. compressirostris, C. curvirostris, C. tshokwe, C. numenius) and their sister taxon Gnathonemus petersii with markedly different snout morphologies and EODs using hybrid capture/HTS DNA metabarcoding of their stomach contents. Our approach allowed for high taxonomic resolution of prey items, including benthic invertebrates, allochthonous invertebrates and vegetation. Comparisons of the diet compositions using quantitative measures and diet overlap indices revealed that all species are able to exploit multiple food niches in their habitats, that is fauna at the bottom, the water surface and the water column. A major part of the diet is larvae of aquatic insects, such as dipterans, coleopterans and trichopterans, known to occur in holes and interstitial spaces of the substrate. The results indicate that different snout morphologies and the associated divergence in the EOD could translate into different prey spectra. This suggests that the diversification in EOD and/or morphology of the feeding apparatus could be under functional adaptation.

A new genome assembly of an African weakly electric fish (Campylomormyrus compressirostris, Mormyridae) indicates rapid gene family evolution in Osteoglossomorpha.

BACKGROUND: Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high-quality genome assembly and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular. RESULTS: A high-quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862 Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A Computational Analysis of gene Family Evolution (CAFE5) comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris. CONCLUSIONS: We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolution, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.

Ontogeny reveals the origin of Gemminger bones in Mormyridae.

Mormyridae are well known and intensively studied for their weak electric organ discharges, which facilitate communication and orientation. The Gemminger bones of Mormyridae are located next to the electrical organ in the caudal peduncle; however, they have not attracted much interest until recently. Therefore, we investigated the diversity of Gemminger bones in mormyrids and studied their ontogenetic development in Mormyrus rume proboscirostris. Gemminger bones are paired, thin, elongated ossifications lying on the dorsal and ventral sides of the caudal peduncle, and usually reach anterior well below the dorsal and anal fin bases. Ontogeny revealed that they are not intermuscular ossifications, as suspected based on the anatomical position of this structure and the systematic position of the mormyrids. Instead, they are membrane ossifications that originate from the fin stays of the dorsal and anal fins.

Intergenus F1-hybrids of African weakly electric fish (Mormyridae: Gnathonemus petersii ♂ × Campylomormyrus compressirostris ♀) are fertile.

Hybridisation is an important element of adaptive radiation in fish but data are limited in weakly electric mormyrid fish in this respect. Recently, it has been shown that intragenus hybrids (Campylomormyrus) are fertile and are able to produce F2-fish. In this paper, we demonstrate that even intergenus hybrids (Gnathonemus petersii ♂ × Campylomormyrus compressirostris ♀) are fertile. Three artificial reproduction (AR) trials, with an average fertilisation rate of ca. 23%, yielded different numbers of survivals (maximally about 50%) of the F1-hybrids. The complete ontogenetic development of these hybrids is described concerning their morphology and electric organ discharge (EOD). Two EOD types emerged at the juvenile stage, which did not change up to adulthood. Type I consisted of four phases and Type II was triphasic. The minimum body length at sexual maturity was between 10 and 11 cm. Malformations, growth and mortality rates are also described.

Evidence for mutual allocation of social attention through interactive signaling in a mormyrid weakly electric fish.

Mormyrid weakly electric fish produce electric organ discharges (EODs) for active electrolocation and electrocommunication. These pulses are emitted with variable interdischarge intervals (IDIs) resulting in temporal discharge patterns and interactive signaling episodes with nearby conspecifics. However, unequivocal assignment of interactive signaling to a specific behavioral context has proven to be challenging. Using an ethorobotical approach, we confronted single individuals of weakly electric Mormyrus rume proboscirostris with a mobile fish robot capable of interacting both physically, on arbitrary trajectories, as well as electrically, by generating echo responses through playback of species-specific EODs, thus synchronizing signals with the fish. Interactive signaling by the fish was more pronounced in response to a dynamic echo playback generated by the robot than in response to playback of static random IDI sequences. Such synchronizations were particularly strong at a distance corresponding to the outer limit of active electrolocation, and when fish oriented toward the fish replica. We therefore argue that interactive signaling through echoing of a conspecific's EODs provides a simple mechanism by which weakly electric fish can specifically address nearby individuals during electrocommunication. Echoing may thus enable mormyrids to mutually allocate social attention and constitute a foundation for complex social behavior and relatively advanced cognitive abilities in a basal vertebrate lineage.

Ontogeny of electric organ and electric organ discharge in Campylomormyrus rhynchophorus (Teleostei: Mormyridae).

The aim of this study was a longitudinal description of the ontogeny of the adult electric organ of Campylomormyrus rhynchophorus which produces as adult an electric organ discharge of very long duration (ca. 25 ms). We could indeed show (for the first time in a mormyrid fish) that the electric organ discharge which is first produced early during ontogeny in 33-mm-long juveniles is much shorter in duration and has a different shape than the electric organ discharge in 15-cm-long adults. The change from this juvenile electric organ discharges into the adult electric organ discharge takes at least a year. The increase in electric organ discharge duration could be causally linked to the development of surface evaginations, papillae, at the rostral face of the electrocyte which are recognizable for the first time in 65-mm-long juveniles and are most prominent at the periphery of the electrocyte.

Intragenus F1-hybrids of African weakly electric fish (Mormyridae: Campylomormyrus tamandua ♂ × C. compressirostris ♀) are fertile.

Hybridization is widespread in fish and constitutes an important mechanism in fish speciation. There is, however, little knowledge about hybridization in mormyrids. F1-interspecies hybrids between Campylomormyrus tamandua ♂ × C. compressirostris ♀ were investigated concerning: (1) fertility; (2) survival of F2-fish and (3) new gene combinations in the F2-generation concerning the structure of the electric organ and features of the electric organ discharge. These F1-hybrids achieved sexual maturity at about 12-13.5 cm total length. A breeding group comprising six males and 13 females spawned 28 times naturally proving these F1-fish to be fertile. On average 228 eggs were spawned, the average fertilization rate was 47.8%. Eggs started to hatch 70-72 h after fertilization, average hatching rate was 95.6%. Average mortality rate during embryonic development amounted to 2.3%. Average malformation rate during the free embryonic stage was 27.7%. Exogenous feeding started on day 11. In total, we raised 353 normally developed larvae all of which died consecutively, the oldest specimen reaching an age of 5 months. During survival, the activities of the larval and adult electric organs were recorded and the structure of the adult electric organ was investigated histologically.

Statistics of Natural Communication Signals Observed in the Wild Identify Important Yet Neglected Stimulus Regimes in Weakly Electric Fish.

Sensory systems evolve in the ecological niches that each species is occupying. Accordingly, encoding of natural stimuli by sensory neurons is expected to be adapted to the statistics of these stimuli. For a direct quantification of sensory scenes, we tracked natural communication behavior of male and female weakly electric fish, Apteronotus rostratus, in their Neotropical rainforest habitat with high spatiotemporal resolution over several days. In the context of courtship, we observed large quantities of electrocommunication signals. Echo responses, acknowledgment signals, and their synchronizing role in spawning demonstrated the behavioral relevance of these signals. In both courtship and aggressive contexts, we observed robust behavioral responses in stimulus regimes that have so far been neglected in electrophysiological studies of this well characterized sensory system and that are well beyond the range of known best frequency and amplitude tuning of the electroreceptor afferents' firing rate modulation. Our results emphasize the importance of quantifying sensory scenes derived from freely behaving animals in their natural habitats for understanding the function and evolution of neural systems.SIGNIFICANCE STATEMENT The processing mechanisms of sensory systems have evolved in the context of the natural lives of organisms. To understand the functioning of sensory systems therefore requires probing them in the stimulus regimes in which they evolved. We took advantage of the continuously generated electric fields of weakly electric fish to explore electrosensory stimulus statistics in their natural Neotropical habitat. Unexpectedly, many of the electrocommunication signals recorded during courtship, spawning, and aggression had much smaller amplitudes or higher frequencies than stimuli used so far in neurophysiological characterizations of the electrosensory system. Our results demonstrate that quantifying sensory scenes derived from freely behaving animals in their natural habitats is essential to avoid biases in the choice of stimuli used to probe brain function.

Disembodying the invisible: electrocommunication and social interactions by passive reception of a moving playback signal.

Mormyrid weakly electric fish have a special electrosensory modality that allows them to actively sense their environment and to communicate with conspecifics by emitting sequences of electric signals. Electroreception is mediated by different types of dermal electroreceptor organs for active electrolocation and electrocommunication, respectively. During electrocommunication, mormyrids exhibit stereotyped discharge sequences and locomotor patterns, which can be induced by playback of electric signals. This raises the question: what sensory information is required to initiate and sustain social interactions, and which electrosensory pathway mediates such interactions? By experimentally excluding stimuli from vision and the lateral line system, we show that Mormyrus rume proboscirostris can rely exclusively on its electrosensory system to track a mobile source of electric communication signals. Detection of electric playback signals induced discharge cessations, followed by double-pulse patterns. The animals tried to interact with the moving signal source and synchronized their discharge activity to the playback. These behaviors were absent in control trials without playback. Silencing the electric organ in some fish did not impair their ability to track the signal source. Silenced fish followed on trajectories similar to those obtained from intact animals, indicating that active electrolocation is no precondition for close-range interactions based on electrocommunication. However, some silenced animals changed their strategy when searching for the stationary playback source, which indicates passive sensing. Social interactions among mormyrids can therefore be induced and mediated by passive reception of electric communication signals without the need for perception of the location of the signal source through other senses.

Electric organ discharge diversification in mormyrid weakly electric fish is associated with differential expression of voltage-gated ion channel genes.

In mormyrid weakly electric fish, the electric organ discharge (EOD) is used for species recognition, orientation and prey localization. Produced in the muscle-derived adult electric organ, the EOD exhibits a wide diversity across species in both waveform and duration. While certain defining EOD characteristics can be linked to anatomical features of the electric organ, many factors underlying EOD differentiation are yet unknown. Here, we report the differential expression of 13 Kv1 voltage-gated potassium channel genes, two inwardly rectifying potassium channel genes, two previously studied sodium channel genes and an ATPase pump in two sympatric species of the genus Campylomormyrus in both the adult electric organ and skeletal muscle. Campylomormyrus compressirostris displays a basal EOD, largely unchanged during development, while C. tshokwe has an elongated, putatively derived discharge. We report an upregulation in all Kv1 genes in the electric organ of Campylomormyrus tshokwe when compared to both skeletal muscle and C. compressirostris electric organ. This pattern of upregulation in a species with a derived EOD form suggests that voltage-gated potassium channels are potentially involved in the diversification of the EOD signal among mormyrid weakly electric fish.

Evidence for Non-neutral Evolution in a Sodium Channel Gene in African Weakly Electric Fish (Campylomormyrus, Mormyridae).

Voltage-gated sodium channels, Nav1, play a crucial role in the generation and propagation of action potentials and substantially contribute to the shape of their rising phase. The electric organ discharge (EOD) of African weakly electric fish (Mormyroidea) is the sum of action potentials fired from all electrocytes of the electric organ at the same time and hence voltage-gated sodium channels are one factor-together with the electrocyte's morphology and innervation pattern-that determines the properties of these EODs. Due to the fish-specific genome duplication, teleost fish possess eight copies of sodium channel genes (SCN), which encode for Nav1 channels. In mormyroids, SCN4aa is solely expressed in the electrocytes of the adult electric organ. In this study, we compared entire SCN4aa sequences of six species of the genus Campylomormyrus and identified nonsynonymous substitutions among them. SCN4aa in Campylomormyrus exhibits a much higher evolutionary rate compared to its paralog SCN4ab, whose expression is not restricted to the electric organ. We also found evidence for strong positive selection on the SCN4aa gene within Mormyridae and along the lineage ancestral to the Mormyridae. We have identified sites at which all nonelectric teleosts are monomorphic in their amino acid, but mormyrids have different amino acids. Our findings confirm the crucial role of SCN4aa in EOD evolution among mormyrid weakly electric fish. The inferred positive selection within Mormyridae makes this gene a prime candidate for further investigation of the divergent evolution of pulse-type EODs among closely related species.

Species delimitation and phylogenetic relationships in a genus of African weakly-electric fishes (Osteoglossiformes, Mormyridae, Campylomormyrus).

African weakly-electric fishes (Mormyridae) are able to communicate through species-specific electric signals; this feature might have favoured the evolutionary radiation observed in this family (over 200 species) by acting as an effective pre-zygotic isolation mechanism. In the present study we used mitochondrial (cytb) and nuclear (rps7, scn4aa) markers in order to reconstruct a species-phylogeny and identify species boundaries for the genus Campylomormyrus, by applying inference methods based on the multispecies coalescent model. Additionally, we employed 16 microsatellite markers, landmark-based morphometric measurements, and electro-physiological analyses as independent lines of evidence to the results obtained from the sequence data. The results show that groups that are morphologically different are also significantly divergent at the genetic level, whereas morphologically similar groups, displaying dissimilar electric signals, do not show enough genetic diversity to be considered separate species. Furthermore, the data confirm the presence of a yet undescribed species within the genus Campylomormyrus.

Cross-tissue and cross-species analysis of gene expression in skeletal muscle and electric organ of African weakly-electric fish (Teleostei; Mormyridae).

BACKGROUND: African weakly-electric fishes of the family Mormyridae are able to produce and perceive weak electric signals (typically less than one volt in amplitude) owing to the presence of a specialized, muscle-derived electric organ (EO) in their tail region. Such electric signals, also known as Electric Organ Discharges (EODs), are used for objects/prey localization, for the identification of conspecifics, and in social and reproductive behaviour. This feature might have promoted the adaptive radiation of this family by acting as an effective pre-zygotic isolation mechanism. Despite the physiological and evolutionary importance of this trait, the investigation of the genetic basis of its function and modification has so far remained limited. In this study, we aim at: i) identifying constitutive differences in terms of gene expression between electric organ and skeletal muscle (SM) in two mormyrid species of the genus Campylomormyrus: C. compressirostris and C. tshokwe, and ii) exploring cross-specific patterns of gene expression within the two tissues among C. compressirostris, C. tshokwe, and the outgroup species Gnathonemus petersii. RESULTS: Twelve paired-end (100 bp) strand-specific RNA-seq Illumina libraries were sequenced, producing circa 330 M quality-filtered short read pairs. The obtained reads were assembled de novo into four reference transcriptomes. In silico cross-tissue DE-analysis allowed us to identify 271 shared differentially expressed genes between EO and SM in C. compressirostris and C.tshokwe. Many of these genes correspond to myogenic factors, ion channels and pumps, and genes involved in several metabolic pathways. Cross-species analysis has revealed that the electric organ transcriptome is more variable in terms of gene expression levels across species than the skeletal muscle transcriptome. CONCLUSIONS: The data obtained indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ's transcriptome; ii) the metabolic activity of the EO might be specialized towards the production and turn-over of membrane structures; iii) several ion channels are highly expressed in the EO in order to increase excitability; iv) several myogenic factors might be down-regulated by transcription repressors in the EO.

Comparative histology of the adult electric organ among four species of the genus Campylomormyrus (Teleostei: Mormyridae).

The electric organ (EO) of weakly electric mormyrids consists of flat, disk-shaped electrocytes with distinct anterior and posterior faces. There are multiple species-characteristic patterns in the geometry of the electrocytes and their innervation. To further correlate electric organ discharge (EOD) with EO anatomy, we examined four species of the mormyrid genus Campylomormyrus possessing clearly distinct EODs. In C. compressirostris, C. numenius, and C. tshokwe, all of which display biphasic EODs, the posterior face of the electrocytes forms evaginations merging to a stalk system receiving the innervation. In C. tamandua that emits a triphasic EOD, the small stalks of the electrocyte penetrate the electrocyte anteriorly before merging on the anterior side to receive the innervation. Additional differences in electrocyte anatomy among the former three species with the same EO geometry could be associated with further characteristics of their EODs. Furthermore, in C. numenius, ontogenetic changes in EO anatomy correlate with profound changes in the EOD. In the juvenile the anterior face of the electrocyte is smooth, whereas in the adult it exhibits pronounced surface foldings. This anatomical difference, together with disparities in the degree of stalk furcation, probably contributes to the about 12 times longer EOD in the adult.

Ontogeny of the electric organs in the electric eel, Electrophorus electricus: physiological, histological, and fine structural investigations.

This study attempts to clarify the controversy regarding the ontogenetic origin of the main organ electrocytes in the electric eel, Electrophorus electricus. The dispute was between an earlier claimed origin from a skeletal muscle precursor [Fritsch, 1881], or from a distinct electrocyte-generating matrix, or germinative zone [Keynes, 1961]. We demonstrate electrocyte formation from a metamerically organized group of pre-electroblasts, splitting off the ventralmost tip of the embryonic trunk mesoderm at the moment of hatching from the egg. We show details of successive stages in the development of rows of electric plates, the electrocytes, by means of conventional histology and electron microscopy. The membrane-bound pre-electroblasts multiply rapidly and then undergo a specific mitosis where they lose their membranes and begin extensive cytoplasm production as electroblasts. Electrical activity, consisting of single and multiple pulses, was noticed in seven-day-old larvae that began to exhibit swimming movements. A separation of discharges into single pulses and trains of higher voltage pulses was seen first in 45-mm-long larvae. A lateralis imus muscle and anal fin ray muscles, implicated by earlier investigators in the formation of electrocytes, begin developing at a time in larval life when eight columns of electrocytes are already present. Axonal innervation is seen very early during electrocyte formation.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae).

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as 'extracystic spermiogenesis'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

Ontogeny of the electric organ discharge and of the papillae of the electrocytes in the weakly electric fish Campylomormyrus rhynchophorus (Teleostei: Mormyridae).

The electric organ of the mormyrid weakly electric fish, Campylomormyrus rhynchophorus (Boulenger, 1898), undergoes changes in both the electric organ discharge (EOD) and the light and electron microscopic morphology as the fish mature from the juvenile to the adult form. Of particular interest was the appearance of papillae, surface specializations of the uninnervated anterior face of the electrocyte, which have been hypothesized to increase the duration of the EOD. In a 24.5 mm long juvenile the adult electric organ (EO) was not yet functional, and the electrocytes lacked papillae. A 40 mm long juvenile, which produced a short biphasic EOD of 1.3 ms duration, shows small papillae (average area 136 µm2 ). In contrast, the EOD of a 79 mm long juvenile was triphasic. The large increase in duration of the EOD to 23.2 ms was accompanied by a small change in size of the papillae (average area 159 µm2 ). Similarly, a 150 mm long adult produced a triphasic EOD of comparable duration to the younger stage (24.7 ms) but featured a prominent increase in size of the papillae (average area 402 µm2 ). Thus, there was no linear correlation between EOD duration and papillary size. The most prominent ultrastructural change was at the level of the myofilaments, which regularly extended into the papillae, only in the oldest specimen-probably serving a supporting function. Physiological mechanisms, like gene expression levels, as demonstrated in some Campylomormyrus species, might be more important concerning the duration of the EOD.