Unraveling the complex phylogeographic history of freshwater fishes in Lower Central America: A study of the electric fish Brachyhypopomus occidentalis.

Lower Central America (LCA) has a complex biogeographic history shaped by the rise of the Isthmus of Panama and the global climatic oscillations of the Pleistocene. These events have been crucial in structuring biodiversity in LCA, but their consequences for the distribution and partitions of genetic diversity across the region remain to be elucidated. We combined complete mitochondrial genomes and nuclear ultraconserved elements (UCEs) to study the phylogeographic history and population genetic structure of the electric fish Brachyhypopomus occidentalis in LCA. Our results are consistent with the known phylogeographic history of B. occidentalis in LCA, but we update this history in several important ways that help illuminate the phylogeographic history of freshwater fishes in the region. We provide: i) support for three waves of colonization, two of which occurred prior to the final closure of the Panama Isthmus; ii) a more precise understanding of each colonization event, with evidence for a larger footprint of the first event, as well as genetic exchange across the continental divide in subsequent events; and iii) evidence for high levels of previously unrecognized population genetic structure across LCA. This updated model of colonization and diversification of B. occidentalis consists of three waves of dispersal and colonization, which triggered the evolution of geographic breaks in both nuclear and mitochondrial genomes across LCA. These processes are tightly linked to the dynamic uplift of the Isthmus, recent volcanic activity in the region, and the sea-level oscillations of the Pleistocene. These results improve previous phylogeographic inferences regarding the distribution and diversification of freshwater fishes in LCA, and generate testable hypotheses to guide future research exploring the factors shaping biodiversity in the region.

The weakly electric fish, Apteronotus albifrons, actively avoids experimentally induced hypoxia.

Anthropogenic environmental degradation has led to an increase in the frequency and prevalence of aquatic hypoxia (low dissolved oxygen concentration, DO), which may affect habitat quality for water-breathing fishes. The weakly electric black ghost knifefish, Apteronotus albifrons, is typically found in well-oxygenated freshwater habitats in South America. Using a shuttle-box design, we exposed juvenile A. albifrons to a stepwise decline in DO from normoxia (> 95% air saturation) to extreme hypoxia (10% air saturation) in one compartment and chronic normoxia in the other. On average, A. albifrons actively avoided the hypoxic compartment below 22% air saturation. Hypoxia avoidance was correlated with upregulated swimming activity. Following avoidance, fish regularly ventured back briefly into deep hypoxia. Hypoxia did not affect the frequency of their electric organ discharges. Our results show that A. albifrons is able to sense hypoxia at non-lethal levels and uses active avoidance to mitigate its adverse effects.

Distribution of the cholinergic nuclei in the brain of the weakly electric fish, Apteronotus leptorhynchus: Implications for sensory processing.

Acetylcholine acts as a neurotransmitter/neuromodulator of many central nervous system processes such as learning and memory, attention, motor control, and sensory processing. The present study describes the spatial distribution of cholinergic neurons throughout the brain of the weakly electric fish, Apteronotus leptorhynchus, using in situ hybridization of choline acetyltransferase mRNA. Distinct groups of cholinergic cells were observed in the telencephalon, diencephalon, mesencephalon, and hindbrain. These included cholinergic cell groups typically identified in other vertebrate brains, for example, motor neurons. Using both in vitro and ex vivo neuronal tracing methods, we identified two new cholinergic connections leading to novel hypotheses on their functional significance. Projections to the nucleus praeeminentialis (nP) arise from isthmic nuclei, possibly including the nucleus lateralis valvulae (nLV) and the isthmic nucleus (nI). The nP is a central component of all electrosensory feedback pathways to the electrosensory lateral line lobe (ELL). We have previously shown that some neurons in nP, TS, and tectum express muscarinic receptors. We hypothesize that, based on nLV/nI cell responses in other teleosts and isthmic connectivity in A. leptorhynchus, the isthmic connections to nP, TS, and tectum modulate responses to electrosensory and/or visual motion and, in particular, to looming/receding stimuli. In addition, we found that the octavolateral efferent (OE) nucleus is the likely source of cholinergic fibers innervating the ELL. In other teleosts, OE inhibits octavolateral hair cells during locomotion. In gymnotiform fish, OE may also act on the first central processing stage and, we hypothesize, implement corollary discharge modulation of electrosensory processing during locomotion.

Plasma proteomic profiles differ between European and North American myotid bats colonized by Pseudogymnoascus destructans.

Emerging fungal diseases have become challenges for wildlife health and conservation. North American hibernating bat species are threatened by the psychrophilic fungus Pseudogymnoascus destructans (Pd) causing the disease called white-nose syndrome (WNS) with unprecedented mortality rates. The fungus is widespread in North America and Europe, however, disease is not manifested in European bats. Differences in epidemiology and pathology indicate an evolution of resistance or tolerance mechanisms towards Pd in European bats. We compared the proteomic profile of blood plasma in healthy and Pd-colonized European Myotis myotis and North American Myotis lucifugus in order to identify pathophysiological changes associated with Pd colonization, which might also explain the differences in bat survival. Expression analyses of plasma proteins revealed differences in healthy and Pd-colonized M. lucifugus, but not in M. myotis. We identified differentially expressed proteins for acute phase response, constitutive and adaptive immunity, oxidative stress defence, metabolism and structural proteins of exosomes and desmosomes, suggesting a systemic response against Pd in North American M. lucifugus but not European M. myotis. The differences in plasma proteomic profiles between European and North American bat species colonized by Pd suggest European bats have evolved tolerance mechanisms towards Pd infection.

Tracking activity patterns of a multispecies community of gymnotiform weakly electric fish in their neotropical habitat without tagging.

Field studies on freely behaving animals commonly require tagging and often are focused on single species. Weakly electric fish generate a species- and individual-specific electric organ discharge (EOD) and therefore provide a unique opportunity for individual tracking without tagging. Here, we present and test tracking algorithms based on recordings with submerged electrode arrays. Harmonic structures extracted from power spectra provide fish identity. Localization of fish based on weighted averages of their EOD amplitudes is found to be more robust than fitting a dipole model. We apply these techniques to monitor a community of three species, Apteronotus rostratus, Eigenmannia humboldtii and Sternopygus dariensis, in their natural habitat in Darién, Panama. We found consistent upstream movements after sunset followed by downstream movements in the second half of the night. Extrapolations of these movements and estimates of fish density obtained from additional transect data suggest that some fish cover at least several hundreds of meters of the stream per night. Most fish, including E. humboldtii, were traversing the electrode array solitarily. From in situ measurements of the decay of the EOD amplitude with distance of individual animals, we estimated that fish can detect conspecifics at distances of up to 2 m. Our recordings also emphasize the complexity of natural electrosensory scenes resulting from the interactions of the EODs of different species. Electrode arrays thus provide an unprecedented window into the so-far hidden nocturnal activities of multispecies communities of weakly electric fish at an unmatched level of detail.

The effect of normoxia exposure on hypoxia tolerance and sensory sampling in a swamp-dwelling mormyrid fish.

Effects of energetic limitations on the performance of sensory systems are generally difficult to quantify. Weakly electric fishes provide an ideal model system to quantify the effects of metabolic stressors on sensory information acquisition, because they use an active-sensing strategy that permits easy measurement of the sensing effort. These fishes discharge an electric signal and sense perturbations of the resulting electric field. We used the mormyrid Petrocephalus degeni to quantify the relationship between routine metabolic rate and the rate of sensory sampling (rate of electric organ discharge, EOD) while under progressive hypoxia by quantifying the critical oxygen tension (PC-MR) and the critical electric organ discharge threshold (PC-EOD). PC-MR was significantly higher in fish acclimated to normoxia for over 40 days compared to animals tested within 1-5 days of capture from a hypoxic swamp, which suggests high costs of maintaining hypoxia tolerance; however, there was no acclimation effect on PC-EOD. All P. degeni reached their PC-EOD prior to their PC-MR. However, below the respective critical tension value, EOD rate decreased more gradually than the metabolic rate suggesting that the fish were increasing the proportion of their energy budget allocated to acquiring sensory information as dissolved-oxygen levels dropped. Trade-offs between sensory sampling and other physiological functions are also suggested by the increase in routine EOD rate with long-term normoxia acclimation, in contrast to metabolic rate, which showed no significant changes. These results highlight the relationship between sensory sampling and metabolic rate in response to progressive hypoxia and the plasticity of hypoxia tolerance.

Effects of hypoxia on aerobic metabolism and active electrosensory acquisition in the African weakly electric fish Marcusenius victoriae.

Environmental hypoxia has effected numerous and well-documented anatomical, physiological and behavioural adaptations in fishes. Comparatively little is known about hypoxia's impacts on sensing because it is difficult to quantify sensory acquisition in vivo. Weakly electric fishes, however, rely heavily on an easily-measurable sensory modality-active electric sensing-whereby individuals emit and detect electric organ discharges (EODs). In this study, hypoxia tolerance of a mormyrid weakly electric fish, Marcusenius victoriae, was assessed by examining both its metabolic and EOD rates using a critical threshold (pcrit ) paradigm. The routine metabolic rate was 1.42 mg O2 h-1 , and the associated critical oxygen tension was 14.34 mmHg. Routine EOD rate was 5.68 Hz with an associated critical tension of 15.14 mmHg. These metabolic indicators of hypoxia tolerance measured in this study were consistent with those in previous studies on M. victoriae and other weakly electric fishes. Furthermore, our results suggest that some aerobic processes may be reduced in favour of maintaining the EOD rate under extreme hypoxia. These findings underscore the importance of the active electrosensory modality to these hypoxia-tolerant fish.

Tempo and mode of allopatric divergence in the weakly electric fish Sternopygus dariensis in the Isthmus of Panama.

Spatial isolation is one of the main drivers of allopatric speciation, but the extent to which spatially-segregated populations accumulate genetic differences relevant to speciation is not always clear. We used data from ultraconserved elements (UCEs) and whole mitochondrial genomes (i.e., mitogenomes) to explore genetic variation among allopatric populations of the weakly electric fish Sternopygus dariensis across the Isthmus of Panama. We found strong genetic divergence between eastern and western populations of S. dariensis. Over 77% of the UCE loci examined were differentially fixed between populations, and these loci appear to be distributed across the species' genome. Population divergence occurred within the last 1.1 million years, perhaps due to global glaciation oscillations during the Pleistocene. Our results are consistent with a pattern of genetic differentiation under strict geographic isolation, and suggest the presence of incipient allopatric species within S. dariensis. Genetic divergence in S. dariensis likely occurred in situ, long after the closure of the Isthmus of Panama. Our study highlights the contribution of spatial isolation and vicariance to promoting rapid diversification in Neotropical freshwater fishes. The study of spatially-segregated populations within the Isthmus of Panama could reveal how genetic differences accumulate as allopatric speciation proceeds.

Mitogenomics of Central American weakly-electric fishes.

Electric fishes are a diverse group of freshwater organisms with the ability to generate electric organ discharges (EODs) that are used for communication and electrolocation. This group (ca. 200 species) has originated in South America, and six species colonized the Central American Isthmus. Here, we assembled the complete mitochondrial genomes (mitogenomes) for three Central American electric fishes (i.e. Sternopygus dariensis, Brachyhypopomus occidentalis, and Apteronotus rostratus), and, based on these data, explored their phylogenetic position among Gymnotiformes. The three mitogenomes show the same gene order, as reported for other fishes, with a size ranging from 16,631 to 17,093 bp. We uncovered a novel 60 bp intergenic spacer (IGS) located between the COII and tRNALys genes, which appears to be unique to the Apteronotidae. Furthermore, phylogenetic relationships supported the traditional monophyly of Gymnotiformes, with the three species positioned within their respective family. In addition, the genus Apteronotus belongs to the early diverging lineage of the order. Finally, we found high sequence divergence (13%) between our B. occidentalis specimen and a sequence previously reported in GenBank, suggesting that the prior mitogenome of B. occidentalis represents a different South American species. Indeed, phylogenetic analyses using Cytochrome b gene across the genus placed the previously reported individual within B. bennetti. Our study provides novel mitogenome resources that will advance our understanding of the diversity and phylogenetic history of Neotropical fishes.

Effects of hypoxia on swimming and sensing in a weakly electric fish.

Low dissolved oxygen (hypoxia) can severely limit fish performance, especially aerobically expensive behaviours including swimming and acquisition of sensory information. Fishes can reduce oxygen requirements by altering these behaviours under hypoxia, but the underlying mechanisms can be difficult to quantify. We used a weakly electric fish as a model system to explore potential effects of hypoxia on swim performance and sensory information acquisition, which enabled us to non-invasively record electric signalling activity used for active acquisition of sensory information during swimming. To quantify potential effects of hypoxia, we measured critical swim speed (Ucrit) and concurrent electric signalling activity under high- and low-dissolved oxygen concentrations in a hypoxia-tolerant African mormyrid fish, Marcusenius victoriae Fish were maintained under normoxia for 6 months prior to experimental treatments, and then acclimated for 8 weeks to normoxia or hypoxia and tested under both conditions (acute: 4 h exposure). Acute hypoxia exposure resulted in a significant reduction in both Ucrit and electric signalling activity in fish not acclimated to hypoxia. However, individuals acclimated to chronic hypoxia were characterized by a higher Ucrit under both hypoxia and normoxia than fish acclimated to normoxia. Following a 6 month re-introduction to normoxia, hypoxia-acclimated individuals still showed increased performance under acute hypoxic test conditions, but not under normoxia. Our results highlight the detrimental effects of hypoxia on aerobic swim performance and sensory information acquisition, and the ability of fish to heighten aerobic performance through acclimation processes that can still influence performance even months after initial exposure.

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.

Pattern of aromatase mRNA expression in the brain of a weakly electric fish, Apteronotus leptorhynchus.

Aromatase is a steroidogenic enzyme involved in the conversion of testosterone into estradiol. Teleosts are unique among vertebrates in possessing two distinct aromatase genes that show different expression patterns within the body. Since the brain is the essential organ underlying the control of behavior, an understanding of the expression pattern of aromatase in the brain can help to identify neural circuits and behaviors that are most likely to be affected by aromatase activity. In addition, identifying species differences in aromatase expression in the brain can further our understanding of divergence in behaviors regulated by local estradiol production and estrogen signaling. Apteronotus leptorhynchus is a species of weakly electric fish in which little is known about sex steroid expression within the brain and its role in electric signaling behavior. The goal of this study was to identify the mRNA expression pattern of aromatase in the brain of A. leptorhynchus. Aromatase mRNA was detected in several parts of the forebrain and in the pituitary gland; however, no aromatase expression was detected in the midbrain or hindbrain. These findings in A. leptorhynchus support a role for aromatase activity in reproduction, but no direct role in electric signaling behavior in non-breeding fish. The findings of this study help to broaden the basis for making phylogenetic comparisons of aromatase expression across teleost lineages as well as different signaling systems, and provide information on behaviors and neural circuits that are potentially affected by local estradiol production in A. leptorhynchus.

Hypoxia acclimation increases novelty response strength during fast-starts in the African mormyrid, Marcusenius victoriae.

Many fishes perform quick and sudden swimming maneuvers known as fast-starts to escape when threatened. In pulse-type weakly electric fishes these responses are accompanied by transient increases in the rate of electric signal production known as novelty responses. While novelty responses may increase an individual's information about their surroundings, they are aerobically powered and may come at a high energetic cost when compared to fast-starts, which rely primarily on anaerobic muscle. The juxtaposition between two key aspects of fast-starts in these fishes - the aerobic novelty response and the anaerobic swimming performance - makes them an interesting model for studying effects of hypoxia on escape performance and sensory information acquisition. We acclimated the hypoxia-tolerant African mormyrid Marcusenius victoriae to either high or low dissolved oxygen (DO) levels for 8weeks, after which fast-starts and novelty responses were quantified under both high (normoxic) and low-DO (hypoxic) test conditions. Hypoxia-acclimated fish exhibited higher maximum curvature than normoxia-acclimated fish. Displacement of normoxia-acclimated fish was not reduced under acute hypoxic test conditions. Novelty responses were given upon each startle, whether or not the fish performed a fast-start; however, novelty responses associated with fast-starts were significantly stronger than those without, suggesting a functional link between fast-start initiation and the motor control of the novelty response. Overall, hypoxia-acclimated individuals produced significantly stronger novelty responses during fast-starts. We suggest that increased novelty response strength in hypoxia-acclimated fish corresponds to an increased rate of sensory sampling, which may compensate for potential negative effects of hypoxia on higher-level processing.

Subsecond Sensory Modulation of Serotonin Levels in a Primary Sensory Area and Its Relation to Ongoing Communication Behavior in a Weakly Electric Fish.

Serotonergic neurons of the raphe nuclei of vertebrates project to most regions of the brain and are known to significantly affect sensory processing. The subsecond dynamics of sensory modulation of serotonin levels and its relation to behavior, however, remain unknown. We used fast-scan cyclic voltammetry to measure serotonin release in the electrosensory system of weakly electric fish, Apteronotus leptorhynchus. These fish use an electric organ to generate a quasi-sinusoidal electric field for communicating with conspecifics. In response to conspecific signals, they frequently produce signal modulations called chirps. We measured changes in serotonin concentration in the hindbrain electrosensory lobe (ELL) with a resolution of 0.1 s concurrently with chirping behavior evoked by mimics of conspecific electric signals. We show that serotonin release can occur phase locked to stimulus onset as well as spontaneously in the ELL region responsible for processing these signals. Intense auditory stimuli, on the other hand, do not modulate serotonin levels in this region, suggesting modality specificity. We found no significant correlation between serotonin release and chirp production on a trial-by-trial basis. However, on average, in the trials where the fish chirped, there was a reduction in serotonin release in response to stimuli mimicking similar-sized same-sex conspecifics. We hypothesize that the serotonergic system is part of an intricate sensory-motor loop: serotonin release in a sensory area is triggered by sensory input, giving rise to motor output, which can in turn affect serotonin release at the timescale of the ongoing sensory experience and in a context-dependent manner.

Burst Firing in the Electrosensory System of Gymnotiform Weakly Electric Fish: Mechanisms and Functional Roles.

Neurons across sensory systems and organisms often display complex patterns of action potentials in response to sensory input. One example of such a pattern is the tendency of neurons to fire packets of action potentials (i.e., a burst) followed by quiescence. While it is well known that multiple mechanisms can generate bursts of action potentials at both the single-neuron and the network level, the functional role of burst firing in sensory processing is not so well understood to date. Here we provide a comprehensive review of the known mechanisms and functions of burst firing in processing of electrosensory stimuli in gymnotiform weakly electric fish. We also present new evidence from existing data showing that bursts and isolated spikes provide distinct information about stimulus variance. It is likely that these functional roles will be generally applicable to other systems and species.

Drift-driven evolution of electric signals in a Neotropical knifefish.

Communication signals are highly diverse traits. This diversity is usually assumed to be shaped by selective forces, whereas the null hypothesis of divergence through drift is often not considered. In Panama, the weakly electric fish Brachyhypopomus occidentalis is widely distributed in multiple independent drainage systems, which provide a natural evolutionary laboratory for the study of genetic and signal divergence in separate populations. We quantified geographic variation in the electric signals of 109 fish from five populations, and compared it to the neutral genetic variation estimated from cytochrome oxidase I (COI) sequences of the same individuals, to test whether drift may be driving divergence of their signals. Signal distances were highly correlated with genetic distances, even after controlling for geographic distances, suggesting that drift alone is sufficient to explain geographic variation in electric signals. Significant differences at smaller geographic scales (within drainages) showed, however, that electric signals may evolve at a faster rate than expected under drift, raising the possibility that additional adaptive forces may be contributing to their evolution. Overall, our data point to stochastic forces as main drivers of signal evolution in this species and extend the role of drift in the evolution of communication systems to fish and electrocommunication.

Predators inhibit brain cell proliferation in natural populations of electric fish, Brachyhypopomus occidentalis.

Compared with laboratory environments, complex natural environments promote brain cell proliferation and neurogenesis. Predators are one important feature of many natural environments, but, in the laboratory, predatory stimuli tend to inhibit brain cell proliferation. Often, laboratory predatory stimuli also elevate plasma glucocorticoids, which can then reduce brain cell proliferation. However, it is unknown how natural predators affect cell proliferation or whether glucocorticoids mediate the neurogenic response to natural predators. We examined brain cell proliferation in six populations of the electric fish, Brachyhypopomus occidentalis, exposed to three forms of predator stimuli: (i) natural variation in the density of predatory catfish; (ii) tail injury, presumably from predation attempts; and (iii) the acute stress of capture. Populations with higher predation pressure had lower density of proliferating (PCNA+) cells, and fish with injured tails had lower proliferating cell density than those with intact tails. However, plasma cortisol did not vary at the population level according to predation pressure or at the individual level according to tail injury. Capture stress significantly increased cortisol, but only marginally decreased cell proliferation. Thus, it appears that the presence of natural predators inhibits brain cell proliferation, but not via mechanisms that depend on changes in basal cortisol levels. This study is the first demonstration of predator-induced alteration of brain cell proliferation in a free-living vertebrate.

Neural maps in the electrosensory system of weakly electric fish.

The active electrosense of weakly electric fish is evolutionarily and developmentally related to passive electrosensation and the lateral line system. It shows the most highly differentiated topographic maps of the receptor array of all these senses. It is organized into three maps in the hindbrain that are, in turn, composed of columns, each consisting of six pyramidal cell classes. The cells in each column have different spatiotemporal processing properties yielding a total of 18 topographic representations of the body surface. The differential filtering by the hindbrain maps is used by superimposed maps in the multi-layered midbrain electrosensory region to extract specific stimulus features related to communication and foraging. At levels beyond the midbrain, topographic mapping of the body surface appears to be lost.

Statistics of the electrosensory input in the freely swimming weakly electric fish Apteronotus leptorhynchus.

The neural computations underlying sensory-guided behaviors can best be understood in view of the sensory stimuli to be processed under natural conditions. This input is often actively shaped by the movements of the animal and its sensory receptors. Little is known about natural sensory scene statistics taking into account the concomitant movement of sensory receptors in freely moving animals. South American weakly electric fish use a self-generated quasi-sinusoidal electric field for electrolocation and electrocommunication. Thousands of cutaneous electroreceptors detect changes in the transdermal potential (TDP) as the fish interact with conspecifics and the environment. Despite substantial knowledge about the circuitry and physiology of the electrosensory system, the statistical properties of the electrosensory input evoked by natural swimming movements have never been measured directly. Using underwater wireless telemetry, we recorded the TDP of Apteronotus leptorhynchus as they swam freely by themselves and during interaction with a conspecific. Swimming movements caused low-frequency TDP amplitude modulations (AMs). Interacting with a conspecific caused additional AMs around the difference frequency of their electric fields, with the amplitude of the AMs (envelope) varying at low frequencies due to mutual movements. Both AMs and envelopes showed a power-law relationship with frequency, indicating spectral scale invariance. Combining a computational model of the electric field with video tracking of movements, we show that specific swimming patterns cause characteristic spatiotemporal sensory input correlations that contain information that may be used by the brain to guide behavior.