Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Simulated predator stimuli reduce brain cell proliferation in two electric fish species, Brachyhypopomus gauderio and Apteronotus leptorhynchus.

The brain structure of many animals is influenced by their predators, but the cellular processes underlying this brain plasticity are not well understood. Previous studies showed that electric fish (Brachyhypopomus occidentalis) naturally exposed to high predator (Rhamdia quelen) density and tail injury had reduced brain cell proliferation compared with individuals facing few predators and those with intact tails. However, these field studies described only correlations between predator exposure and cell proliferation. Here, we used a congener Brachyhypopomus gauderio and another electric fish Apteronotus leptorhynchus to experimentally test the hypothesis that exposure to a predator stimulus and tail injury causes alterations in brain cell proliferation. To simulate predator exposure, we either amputated the tail followed by short-term (1 day) or long-term (17-18 days) recovery or repeatedly chased intact fish with a plastic rod over a 7 day period. We measured cell proliferation (PCNA+ cell density) in the telencephalon and diencephalon, and plasma cortisol, which commonly mediates stress-induced changes in brain cell proliferation. In both species, either tail amputation or simulated predator chase decreased cell proliferation in the telencephalon in a manner resembling the effect of predators in the field. In A. leptorhynchus, cell proliferation decreased drastically in the short term after tail amputation and partially rebounded after long-term recovery. In B. gauderio, tail amputation elevated cortisol levels, but repeated chasing had no effect. In A. leptorhynchus, tail amputation elevated cortisol levels in the short term but not in the long term. Thus, predator stimuli can cause reductions in brain cell proliferation, but the role of cortisol is not clear.

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.

The energetics of electric organ discharge generation in gymnotiform weakly electric fish.

Gymnotiform weakly electric fish produce an electric signal to sense their environment and communicate with conspecifics. Although the generation of such relatively large electric signals over an entire lifetime is expected to be energetically costly, supporting evidence to date is equivocal. In this article, we first provide a theoretical analysis of the energy budget underlying signal production. Our analysis suggests that wave-type and pulse-type species invest a similar fraction of metabolic resources into electric signal generation, supporting previous evidence of a trade-off between signal amplitude and frequency. We then consider a comparative and evolutionary framework in which to interpret and guide future studies. We suggest that species differences in signal generation and plasticity, when considered in an energetics context, will not only help to evaluate the role of energetic constraints in the evolution of signal diversity but also lead to important general insights into the energetics of bioelectric signal generation.

Energetic cost of communication.

Communication signals may be energetically expensive or inexpensive to produce, depending on the function of the signal and the competitive nature of the communication system. Males of sexually selected species may produce high-energy advertisement signals, both to enhance detectability and to signal their size and body condition. Accordingly, the proportion of the energy budget allocated to signal production ranges from almost nothing for many signals to somewhere in excess of 50% for acoustic signals in short-lived sexually selected species. Recent data from gymnotiform electric fish reveal mechanisms that regulate energy allocated to sexual advertisement signals through dynamical remodeling of the excitable membranes in the electric organ. Further, males of the short-lived sexually selected species, Brachyhypopomus gauderio, trade off among different metabolic compartments, allocating energy to signal production while reducing energy used in other metabolic functions. Female B. gauderio, by contrast, do not trade off energy between signaling and other functions. To fuel energetically expensive signal production, we expect a continuum of strategies to be adopted by animals of different life history strategies. Future studies should explore the relation between life history and energy allocation trade-offs.

Social competition affects electric signal plasticity and steroid levels in the gymnotiform fish Brachyhypopomus gauderio.

Sexually-selected communication signals can be used by competing males to settle contests without incurring the costs of fighting. Steroid regulation of these signals can render them as reliable indicators of a male's physiological state. We investigated how plasticity in electrocommunication signals is driven by social competition for mates, mediated by steroid hormones, and subject to the effects of past social experience. We measured the electric waveform's amplitude and duration and steroid hormone levels of male gymnotiform electric fish (Brachyhypopomus gauderio) following week-long periods of social isolation, and low or high social competition. To quantify the effect of social history on the modulation of the electric signal, six groups of six males experienced all three social conditions but in different order. We found that males differentially modulate their electric signals depending on the order they experienced these conditions. Thus, past social interactions affect both present and future social electric signals. Cortisol levels and the amplitude of the electric signal appeared to track the intensity of competition, while androgen levels and the duration of the electric signal only responded to the presence (low and high competition) or absence (isolation) of a social environment (low and high androgens respectively). In addition, cortisol levels were related to the body size of the males at high social competition. Taken together, these findings suggest that the capacity of males to modulate their signals in response to social competition is regulated by steroids.

Sex differences in energetic costs explain sexual dimorphism in the circadian rhythm modulation of the electrocommunication signal of the gymnotiform fish Brachyhypopomus pinnicaudatus.

To understand the evolution of sexually dimorphic communication signals, we must quantify their costs, including their energetic costs, the regulation of these costs, and the difference between the costs for the sexes. Here, we provide the first direct measurements of the relative energy expended on electric signals and show for the focal species Brachyhypopomus pinnicaudatus that males spend a significantly greater proportion of their total energy budget on signal generation (11-22%) compared with females (3%). Both sexes significantly reduce the energy spent on electric signals during daylight hours through circadian modulation of the amplitude, duration and repetition rate of the electric signal, but this effect is more marked in males. Male body condition predicted the energy spent on electric signals (R(2)=0.75). The oxygen consumed by males for signal production closely paralleled the product of the electric signal's waveform area (R(2)=0.99) and the discharge rate (R(2)=0.59), two signal parameters that can be assessed directly by conspecifics. Thus the electric communication signal of males carries the information to reveal their body condition to prospective mates and competing males. Because the electric signal constitutes a significant fraction of the energy budget, energy savings, along with predation avoidance, provides an adaptive basis for the production of circadian rhythms in electric signals.

Opposing actions of 5HT1A and 5HT2-like serotonin receptors on modulations of the electric signal waveform in the electric fish Brachyhypopomus pinnicaudatus.

Serotonin (5-HT) is an indirect modulator of the electric organ discharge (EOD) in the weakly electric gymnotiform fish, Brachyhypopomus pinnicaudatus. Injections of 5-HT enhance EOD waveform "masculinity", increasing both waveform amplitude and the duration of the second phase. This study investigated the pharmacological identity of 5-HT receptors that regulate the electric waveform and their effects on EOD amplitude and duration. We present evidence that two sets of serotonin receptors modulate the EOD in opposite directions. We found that the 5HT1AR agonist 8-OH-DPAT diminishes EOD duration and amplitude while the 5HT1AR antagonist WAY100635 increases these parameters. In contrast, the 5HT2R agonist alpha-Me-5-HT increases EOD amplitude but not duration, yet 5-HT-induced increases in EOD duration can be inhibited by blocking 5HT2A/2C-like receptors with ketanserin. These results show that 5-HT exerts bi-directional control of EOD modulations in B. pinnicaudatus via action at receptors similar to mammalian 5HT1A and 5HT2 receptors. The discordant amplitude and duration response suggests separate mechanisms for modulating these waveform parameters.

Circadian rhythms in electric waveform structure and rate in the electric fish Brachyhypopomus pinnicaudatus.

Weakly electric fish have long been known to express day-night oscillations in their discharge rates, and in the amplitude and duration of individual electric organ discharges (EODs). Because these oscillations are altered by social environment and neuroendocrine interactions, electric fish are excellent organisms for exploring the social and neuroendocrine regulation of circadian rhythm expression. Previous studies asserting that these oscillations are circadian rhythms have been criticized for failing to control temperature and randomize feeding regimes, or for running the fish under constant conditions for just 2-3 days. Here we show that the day-night oscillations in the EODs of the neotropical gymnotiform fish Brachyhypopomus pinnicaudatus free-run for over a week under constant photic and thermal conditions, and randomized food provisioning. Sex differences were apparent in strength and magnitude of the circadian oscillations; male oscillations were stronger and larger. All three parameters retain a common oscillation period while differing in the persistence of oscillation strength and magnitude, a difference consistent with proposals by others that declines of behavioral circadian rhythms may result from breakdowns downstream of the central oscillator.

Serotonin modulates the electric waveform of the gymnotiform electric fish Brachyhypopomus pinnicaudatus.

The gymnotiform electric fish Brachyhypopomus pinnicaudatus communicates with a sexually dimorphic electric waveform, the electric organ discharge (EOD). Males display pronounced circadian rhythms in the amplitude and duration of their EODs. Changes in the social environment influence the magnitudes of these circadian rhythms and also produce more transient responses in the EOD waveforms. Here we show that injections of serotonin produce quick, transient, dose-dependent enhancements of the male EOD characters similar to those induced by encounters with another male. The response to serotonin administered peripherally begins 5-10 min post injection and lasts approximately 3 h. The magnitude of the response to serotonin is tightly associated with the magnitude of the day-to-night swing of the circadian rhythm prior to injection. Taken together these findings suggest that the male's social environment influences his response to serotonin by altering the function of some part of the downstream chain between the serotonin receptors and the ion channels involved in control of the EOD waveform. Although chronic activation of serotonin circuitry is widely known to elicit subordinate behavior, we find that 5-HT initially increases a dominance signal in these fish. These findings are consistent with the emerging view that serotonin facilitates different adaptive responses to acute and chronic social challenge and stress.