Laura Quintana

Biogeography and Breeding in Gymnotiformes from Uruguay

This study was carried out in Uruguay (30–35°S), South America, with two complementary approaches. First, an extensive exploration of Uruguayan freshwaters allowed us to assess the distribution of the two major species of Gymnotiformes (out of 4) across sites. Gymnotus carapo was uniformly distributed in Uruguayan territory, whereas Brachyhypopomus pinnicaudatus was observed in the northern and eastern part of the country. There was a highly significant negative correlation between the relative abundance of Brachyhypopomus pinnicaudatus and pH and water conductivity. Moreover, these environmental factors are significant contributors to its spatial differences in relative abundance. Second, temperature, conductivity, photoperiod, and the structure of a Brachyhypopomus pinnicaudatus population were analyzed across seasons in a small lake over a two-year period. Water temperature and photoperiod exhibited important seasonal changes, whereas water conductivity remained low and relatively constant. The presence of sexually mature males, females, and the sudden increase of juveniles indicated the occurrence of the breeding season in November, December, and January, coinciding with high mean water temperatures and extreme photoperiod. These results agree with previous data that support the hypothesis of temperature as an important environmental factor for the onset of breeding in Gymnotiformes from the temperate zone.

Water temperature sensitivity of EOD waveform in Brachyhypopomus pinnicaudatus

Abstract The mechanisms that trigger the onset of the breeding season depend on geographical latitude. At the edge of Gymnotiform distribution in America, variations in day length and water temperature are likely cues to initiate breeding. In this study we aim to clarify the role of temperature and the interaction between temperature and hormonal state upon electric organ discharge waveform. In breeding ponds, we measured naturally occurring changes of water temperature and of electric organ discharge waveform during two 48-h periods in a sample of identified mature males and females of Brachyhypopomus pinnicaudatus. Water temperature, day-night cycle, and sexual maturity each modified electric organ discharge waveform. Temperature sensitivity was also evaluated in the laboratory in adult sexually-differentiated individuals, adult non-differentiated fish, juveniles, and testosterone-treated fish. Our data strongly suggest an interaction between the effects of temperature and steroid hormones upon electric organ discharge waveform. High temperature (30 °C) induced a significant decay of head negative phase amplitude in temperature-sensitive fish. This sensitivity was observed in physiological conditions that coincide with low levels of steroid hormones: juveniles and adult fish kept in captivity at 20–21 °C. Conversely, temperature resistance was observed in mature fish in the breeding habitat and was induced by testosterone treatment and by captivity at 27–28 °C.

Temperature induces gonadal maturation and affects electrophysiological sexual maturity indicators in Brachyhypopomus pinnicaudatus from a temperate climate.

SUMMARY In contrast to most of the previous studies in gymnotiform reproduction, which have been conducted in the tropical region, this study examines a gymnotid from the temperate region in both the natural habitat and the laboratory. The gonadal histology of Brachyhypopomus pinnicaudatus is described for the first time. The male had a paired, lobular testis of the unrestricted spermatogonial type, and females a paired saccular cystovary. Analysis of gonads and their annual cycle enabled us to confirm the breeding season and to conclude that this species is a multiple spawner. Water temperature and photoperiod showed the expected annual cycles for the region. High temperature and a 14 h:10 h L:D photoperiod in the natural habitat coincided with (1) mature gonadal stages, (2) electrophysiological sexual dimorphism: males present a lengthened negative phase in their electric organ discharge (EOD) and (3) decreased temperature sensitivity of the EOD: the waveform does not change when temperature increases above 20°C. Acclimation to sustained high temperature (30 days, 28°C, 12 h:12 h L:D, low conductivity) induced gonad maturation along with EOD dimorphism. Our data show that high environmental temperature is enough to trigger sexual maturity in Brachyhypopomus pinnicaudatus from a temperate climate.

Environmental and hormonal influences upon EOD waveform in gymnotiform pulse fish.

Temperature is a major variable that affects all biological systems. Environmental temperature determines animal geographical distribution and activity, and influences their reproductive cycle, particularly within the temperate zone. Temperature, as a physical parameter, also strongly affects excitable tissues. The hypothesis of temperature as the most important environmental cue for the onset of breeding in gymnotiform pulse fish of the temperate zone is supported by: (a) a clear temporal correlation that was observed in the wild between water temperature and sexual maturity, and (b) the induction of gonadal maturation and sexual differences after acclimation at high temperature (28 degrees C) in the laboratory. Temperature sensitivity of EOD waveform (described in Brachyhypopomus pinnicaudatus and Gymnotus carapo) is characterized by the decrease of the EODs late head-negative phase as temperature increases. This phenomenon depends on electrocyte properties since: (a) experimentally induced changes of discharge rate at constant temperature generate smaller EOD distortion, and (b) the effect of temperature upon EOD also depends on water conductivity. Temperature sensitivity of EOD waveform is negatively correlated with gonadal maturity in Brachyhypopomus pinnicaudatus. High temperature sensitivity was observed during the non-breeding season, whereas low temperature sensitivity was recorded during the breeding season. Temperature sensitivity of EOD waveform in both Brachyhypopomus pinnicaudatus and Gymnotus carapo was modulated by: (a) testosterone treatment (100 microg/g) that decreased temperature sensitivity, and (b) acclimation at high temperature (28 degrees C, 1 month) that also decreased temperature sensitivity. Temperature is probably acting through the neuroendocrine system, and ultimately interacting with steroid hormones in their effects upon EOD waveform.

Brain androgen receptor expression correlates with seasonal changes in the behavior of a weakly electric fish, Brachyhypopomus gauderio

Seasonal breeders are superb models for understanding natural relationships between reproductive behavior and its neural bases. We investigated the cellular bases of hormone effects in a weakly pulse-type electric fish with well-defined hormone-sensitive communication signals. Brachyhypopomus gauderio males emit social electric signals (SESs) consisting of rate modulations of the electric organ discharge during the breeding season. This discharge is commanded by a medullary pacemaker nucleus (PN), composed of pacemaker and relay neurons. We analyzed the contribution of androgen receptor (AR) expression to the seasonal generation of SESs, by examining the presence of ARs in the PN in different experimental groups: breeding, non-breeding, and testosterone (T)-implanted non-breeding males. AR presence and distribution in the CNS was assessed through western blotting and immunohistochemistry using the PG-21 antibody, which was raised against the human AR. We found AR immunoreactivity, for the first time in a pulse-type Gymnotiform, in several regions throughout the brain. In particular, this is the first report to reveal the presence of AR in both pacemaker and relay neurons within the Gymnotiform PN. The AR immunoreactivity was present in breeding males and could be induced in T-implanted non-breeding males. This seasonal and T-induced AR expression in the PN suggests that androgens may play an important role in the generation of SESs by modulating intrinsic electrophysiological properties of pacemaker and relay neurons.

Sexual and seasonal plasticity in the emission of social electric signals. Behavioral approach and neural bases

Behavior in electric fish includes modulations of a stereotyped electric organ discharge (EOD) in addition to locomotor displays. Gymnotiformes can modulate the EOD rate to produce signals that participate in different behaviors. We studied the reproductive behavior of Brachyhypopomus pinnicaudatus both in the wild and laboratory settings. During the breeding season, fish produce sexually dimorphic social electric signals (SES): males emit three types of chirps (distinguished by their duration and internal structure), and accelerations, whereas females interrupt their EOD. Since these SES imply EOD frequency modulations, the pacemaker nucleus (PN) is involved in their generation and constitutes the main target organ to explore seasonal and sexual plasticity of the CNS. The PN has two types of neurons, pacemakers and relays, which receive modulatory inputs from pre-pacemaker structures. These neurons show an anisotropic rostro-caudal and dorso-ventral distribution that is paralleled by different field potential waveforms in distinct portions of the PN. In vivo glutamate injections in different areas of the PN provoke different kinds of EOD rate modulations. Ventral injections produce chirp-like responses in breeding males and EOD interruptions in breeding females, whereas dorsal injections provoke EOD frequency rises in both sexes. In the non-breeding season, males and females respond with interruptions when stimulated ventrally and frequency rises when injected dorsally. Our results show that changes of glutamate effects in the PN could explain the seasonal and sexual differences in the generation of SES. By means of behavioral recordings both in the wild and in laboratory settings, and by electrophysiological and pharmacological experiments, we have identified sexual and seasonal plasticity of the CNS and explored its underlying mechanisms.

A central pacemaker that underlies the production of seasonal and sexually dimorphic social signals: functional aspects revealed by glutamate stimulation

The cyclic enrichment of behavioral repertoires is a common event in seasonal breeders. Breeding males Brachyhypopomus gauderio produce electric organ discharge (EOD) rate modulations called chirps while females respond with interruptions. The electromotor system is commanded by a pacemaker nucleus (PN) which sets the basal rate and produces the rate modulations. We focused on identifying functional, seasonal and sexual differences in this nucleus in correlation to these differences in behavior. The in vivo response to glutamate injection in the PN was seasonal, sexually dimorphic and site specific. Non-breeding adults and breeding females injected in dorsal and ventral sites generated EOD rate increases and interruptions, respectively. Reproductive males added a conspicuous communication signal to this repertoire. They chirped repetitively when we injected glutamate in a very restricted area of the ventral–rostral nucleus, surprisingly one with a low number of relay cell somata. This study shows that the PN is functionally organized in regions in a caudal–rostral axis, besides the previously documented dorsal–ventral division. Functional regions are revealed by seasonal changes that annually provide this nucleus with the cellular mechanisms that allow the bursting activity underlying chirp production, only in males.

A central pacemaker that underlies the production of seasonal and sexually dimorphic social signals: anatomical and electrophysiological aspects.

Our long-term goal is to approach the understanding of the anatomical and physiological bases for communication signal diversity in gymnotiform fishes as a model for vertebrate motor pattern generation. Brachyhypopomus gauderio emits, in addition to its electric organ discharge (EOD) at basal rate, a rich repertoire of rate modulations. We examined the structure of the pacemaker nucleus, responsible for the EOD rate, to explore whether its high output signal diversity was correlated to complexity in its neural components or regional organization. We confirm the existence of only two neuron types and show that the previously reported dorsal–caudal segregation of these neurons is accompanied by rostral–caudal regionalization. Pacemaker cells are grouped dorsally in the rostral half of the nucleus, and relay cells are mainly ventral and more abundant in the caudal half. Relay cells are loosely distributed from the center to the periphery of the nucleus in correlation to somata size. Our findings support the hypothesis that regional organization enables a higher diversity of rate modulations, possibly offering distinct target areas to modulatory inputs. Since no anatomical or electrophysiological seasonal or sexual differences were found, we explored these aspects from a functional point of view in a companion article.

Local vasotocin modulation of the pacemaker nucleus resembles distinct electric behaviors in two species of weakly electric fish

The neural bases of social behavior diversity in vertebrates have evolved in close association with hypothalamic neuropeptides. In particular, arginine-vasotocin (AVT) is a key integrator underlying differences in behavior across vertebrate taxa. Behavioral displays in weakly electric fish are channeled through specific patterns in their electric organ discharges (EODs), whose rate is ultimately controlled by a medullary pacemaker nucleus (PN). We first explored interspecific differences in the role of AVT as modulator of electric behavior in terms of EOD rate between the solitary Gymnotus omarorum and the gregarious Brachyhypopomus gauderio. In both species, AVT IP injection (10μg/gbw) caused a progressive increase of EOD rate of about 30%, which was persistent in B. gauderio, and attenuated after 30min in G. omarorum. Secondly, we demonstrated by in vitro electrophysiological experiments that these behavioral differences can be accounted by dissimilar effects of AVT upon the PN in itself. AVT administration (1μM) to the perfusion bath of brainstem slices containing the PN produced a small and transient increase of PN activity rate in G. omarorum vs the larger and persistent increase previously reported in B. gauderio. We also identified AVT neurons, for the first time in electric fish, using immunohistochemistry techniques and confirmed the presence of hindbrain AVT projections close to the PN that might constitute the anatomical substrate for AVT influences on PN activity. Taken together, our data reinforce the view of the PN as an extremely plastic medullary central pattern generator that not only responds to higher influences to adapt its function to diverse contexts, but also is able to intrinsically shape its response to neuropeptide actions, thus adding a hindbrain target level to the complexity of the global integration of central neuromodulation of electric behavior.

Extra-gonadal steroids modulate non-breeding territorial aggression in weakly electric fish.

The neuroendocrine control of intraspecific aggression is a matter of current debate. Although aggression in a reproductive context has been associated with high levels of circulating androgens in a broad range of species, it has also been shown to occur during the non-breeding season when gonads are regressed and plasma steroid hormone levels are low. In mammals and birds the aromatization of androgens into estrogens plays a key role in the regulation of aggression in both the breeding and non-breeding seasons. This is the first study in a teleost fish to explore the role of steroids in the modulation of non-breeding aggression. Gymnotus omarorum is a highly aggressive teleost fish that exhibits aggression all year-round. We analyzed male-male non-breeding agonistic behavior, compared circulating 11-Ketotestosterone (11-KT) levels between dominants and isolated males, assessed the regulatory role of aromatization of androgens into estrogens, and evaluated the gonads as a source of these sex steroids. We found that high levels of aggression occurred in the non-breeding season despite low plasma 11-KT levels, and that there was no difference in 11-KT levels between dominant and isolated males. We demonstrated that acute aromatase inhibition decreased aggression, distorted contest dynamics, and affected expected outcome. We also found that castrated individuals displayed aggressive behavior indistinguishable from non-castrated males. Our results show, for the first time in teleost fish, that territorial aggression of G. omarorum during the non-breeding season depends on a non-gonadal estrogenic pathway.