Lisa A. Mangiamele

Acoustic modulation of immediate early gene expression in the auditory midbrain of female túngara frogs

To better understand the molecular consequences of auditory processing in frogs, we investigated the acoustic modulation of two immediate early genes (IEGs), egr-1 and fos, in the auditory midbrain of female túngara frogs. Since túngara frog egr-1 had already been identified, we first isolated a túngara-specific fos clone using degenerate PCR followed by Rapid Amplification of cDNA Ends. In order to examine the temporal kinetics of acoustically modulated IEG mRNA expression, we first acoustically isolated females collected from a mating chorus and analyzed the decline in IEG expression in the torus semicircularis (homolog of the inferior colliculus). We found that IEG mRNA levels declined rapidly and reached baseline within 2 h. Next, we presented females with a 30-min recording of a mating chorus and analyzed IEG expression following different survival times. We found that IEG expression increased within 15-30 min of sound presentation but, compared to other vertebrates, in the túngara frog it took longer to reach the highest and lowest mRNA levels in response to sound and isolation, respectively. We also found that acoustic stimulation of egr-1 and fos differed in the three subdivisions of the torus semicircularis, suggesting that, as in birds, the two genes could provide largely different information when used in IEG mapping studies. While our results confirm the generality of sensory-induced IEG expression in vertebrates, whether the longer time course of IEG expression that we observed represents a species difference in the mechanisms of IEG transcription awaits further study.

Neural Activity Patterns in Response to Interspecific and Intraspecific Variation in Mating Calls in the Tungara Frog

Background During mate choice, individuals must classify potential mates according to species identity and relative attractiveness. In many species, females do so by evaluating variation in the signals produced by males. Male túngara frogs (Physalaemus pustulosus) can produce single note calls (whines) and multi-note calls (whine-chucks). While the whine alone is sufficient for species recognition, females greatly prefer the whine-chuck when given a choice. Methodology/Principal Findings To better understand how the brain responds to variation in male mating signals, we mapped neural activity patterns evoked by interspecific and intraspecific variation in mating calls in túngara frogs by measuring expression of egr-1. We predicted that egr-1 responses to conspecific calls would identify brain regions that are potentially important for species recognition and that at least some of those brain regions would vary in their egr-1 responses to mating calls that vary in attractiveness. We measured egr-1 in the auditory brainstem and its forebrain targets and found that conspecific whine-chucks elicited greater egr-1 expression than heterospecific whines in all but three regions. We found no evidence that preferred whine-chuck calls elicited greater egr-1 expression than conspecific whines in any of eleven brain regions examined, in contrast to predictions that mating preferences in túngara frogs emerge from greater responses in the auditory system. Conclusions Although selectivity for species-specific signals is apparent throughout the túngara frog brain, further studies are necessary to elucidate how neural activity patterns vary with the attractiveness of conspecific mating calls.

Testosterone rapidly increases ejaculate volume and sperm density in competitively breeding goldfish through an estrogenic membrane receptor mechanism

The social environment can have dramatic influences on reproductive behavior and physiology in many vertebrate species. In males, interactions with conspecifics affect physiological processes that increase an individuals ability to compete for mates. For example, in some species, males rapidly adjust the number of sperm they ejaculate in response to sociosexual cues from male and female conspecifics, however, little is known about the physiological mechanisms mediating this behavior. In goldfish, as in many vertebrates, social cues also drive transient surges of the gonadal hormone testosterone (T), which induces rapid effects on cellular processes via its conversion to estradiol (E2). We asked whether such surges rapidly influence ejaculate quantity and quality by experimentally manipulating peripheral levels of T and E2. We show that male goldfish injected with T increased ejaculate (milt) volume and sperm density within just 1 hr. Furthermore, increases in expressible milt were dependent on the conversion of T to E2 by the enzyme aromatase, required activation of estrogen receptors α and β, and were also elicited by BSA-conjugated E2, which acts on cell membrane-bound estrogen receptors. Together, these findings represent a novel steroid mechanism for the social modulation of sperm output over the short time scales that characterize reproductive encounters, and thus demonstrate a previously undescribed functional consequence of rapid estrogen signaling mechanisms. We suggest that such mechanisms may play a critical role in the enhancement of physiological and behavioral processes that increase reproductive success in competitive mating contexts.

GPER/GPR30, a membrane estrogen receptor, is expressed in the brain and retina of a social fish (Carassius auratus) and colocalizes with isotocin

Estradiol rapidly (within 30 minutes) influences a variety of sociosexual behaviors in both mammalian and nonmammalian vertebrates, including goldfish, in which it rapidly stimulates approach responses to the visual cues of females. Such rapid neuromodulatory effects are likely mediated via membrane‐associated estrogen receptors; however, the localization and distribution of such receptors within the nervous system is not well described. To begin to address this gap, we identified GPER/GPR30, a G‐protein‐coupled estrogen receptor, in goldfish (Carassius auratus) neural tissue and used reverse‐transcription polymerase chain reaction (RT‐PCR) and in situ hybridization to test if GPR30 is expressed in the brain regions that might mediate visually guided social behaviors in males. We then used immunohistochemistry to determine whether GPR30 colocalizes with isotocin‐producing cells in the preoptic area, a critical node in the highly conserved vertebrate social behavior network. We used quantitative (q)PCR to test whether GPR30 mRNA levels differ in males in breeding vs. nonbreeding condition and in males that were socially interacting with a female vs. a rival male. Our results show that GPR30 is expressed in the retina and in many brain regions that receive input from the retina and/or optic tectum, as well as in a few nodes in the social behavior network, including cell populations that produce isotocin. J. Comp. Neurol. 525:252–270, 2017.

Increased androgenic sensitivity in the hind limb muscular system marks the evolution of a derived gestural display

Significance Diverse species signal using limb gestures, but little is known about how selection incorporates such movements into display routines. We study this issue in a tropical frog that produces complex waving displays with its hind limbs. We find not only that androgenic hormones activate such signaling behavior, but also that the signal’s recent evolution is marked by a dramatic increase in androgenic sensitivity of the thigh muscles that control hind limb maneuvering. Moreover, we demonstrate that this muscular phenotype mirrors that which is found in the larynx of other frogs that primarily produce androgen-dependent vocalizations as social signals. We therefore uncover strong coevolution between the emergence of complex sexual gestural signals and enhanced androgenic signaling mechanisms in the muscular system. Physical gestures are prominent features of many species’ multimodal displays, yet how evolution incorporates body and leg movements into animal signaling repertoires is unclear. Androgenic hormones modulate the production of reproductive signals and sexual motor skills in many vertebrates; therefore, one possibility is that selection for physical signals drives the evolution of androgenic sensitivity in select neuromotor pathways. We examined this issue in the Bornean rock frog (Staurois parvus, family: Ranidae). Males court females and compete with rivals by performing both vocalizations and hind limb gestural signals, called “foot flags.” Foot flagging is a derived display that emerged in the ranids after vocal signaling. Here, we show that administration of testosterone (T) increases foot flagging behavior under seminatural conditions. Moreover, using quantitative PCR, we also find that adult male S. parvus maintain a unique androgenic phenotype, in which androgen receptor (AR) in the hind limb musculature is expressed at levels ∼10× greater than in two other anuran species, which do not produce foot flags (Rana pipiens and Xenopus laevis). Finally, because males of all three of these species solicit mates with calls, we accordingly detect no differences in AR expression in the vocal apparatus (larynx) among taxa. The results show that foot flagging is an androgen-dependent gestural signal, and its emergence is associated with increased androgenic sensitivity within the hind limb musculature. Selection for this novel gestural signal may therefore drive the evolution of increased AR expression in key muscles that control signal production to support adaptive motor performance.

The geomagnetic environment in which sea turtle eggs incubate affects subsequent magnetic navigation behaviour of hatchlings

Loggerhead sea turtle hatchlings (Caretta caretta) use regional magnetic fields as open-ocean navigational markers during trans-oceanic migrations. Little is known, however, about the ontogeny of this behaviour. As a first step towards investigating whether the magnetic environment in which hatchlings develop affects subsequent magnetic orientation behaviour, eggs deposited by nesting female loggerheads were permitted to develop in situ either in the natural ambient magnetic field or in a magnetic field distorted by magnets placed around the nest. In orientation experiments, hatchlings that developed in the normal ambient field oriented approximately south when exposed to a field that exists near the northern coast of Portugal, a direction consistent with their migratory route in the northeastern Atlantic. By contrast, hatchlings that developed in a distorted magnetic field had orientation indistinguishable from random when tested in the same north Portugal field. No differences existed between the two groups in orientation assays involving responses to orbital movements of waves or sea-finding, neither of which involves magnetic field perception. These findings, to our knowledge, demonstrate for the first time that the magnetic environment present during early development can influence the magnetic orientation behaviour of a neonatal migratory animal.

Acoustically Evoked Immediate Early Gene Expression in the Pallium of Female Túngara Frogs

In anurans, much is known about the role of the auditory midbrain in processing conspecific calls, but comparatively little is known about the role of the pallium. To address this deficiency, we investigated the induction of the immediate early gene egr-1 by natural mate chorus in the medial, dorsal, lateral, and ventral pallium of female túngara frogs. We found strong acoustically evoked egr-1 expression in the dorsal medial pallium (p < 0.01) and ventral pallium (p = 0.02), with a weaker effect in the lateral pallium (p = 0.05). In the ventral pallium, acoustically induced egr-1 expression was stronger in the anterior portion. Measures of movement and olfactory activity could not explain a significant portion of acoustically evoked pallial egr-1 expression. In contrast, egr-1 expression in the auditory midbrain covaried with egr-1 expression in the dorsal medial pallium and ventral pallium, suggesting that their activity was coupled with auditory activity. Taken together, these results suggest that the acoustically evoked egr-1 expression in the dorsal medial pallium and ventral pallium were a direct result of auditory stimulation. Furthermore, although both anatomical and electrophysiological evidence demonstrate that multiple modalities overlap in the frog pallium, our results show that a multimodal stimulus is not required to activate pallial neurons. Although the functional role of the frog pallium is not known, our results demonstrate that species-specific sounds activate spatially segregated and anatomically distinct areas of the frog pallium, inviting further investigation into the role of the frog pallium in acoustic communication.

Pheromone exposure influences preoptic arginine vasotocin gene expression and inhibits social approach behavior in response to rivals but not potential mates.

The nonapeptides arginine vasotocin (AVT) and vasopressin mediate a variety of social behaviors in vertebrates. However, the effects of these peptides on behavior can vary considerably both between and within species. AVT, in particular, stimulates aggressive and courtship responses typical of dominant males in several species, although it can also inhibit social interactions in some cases. Such differential effects may depend upon AVT influences within brain circuits that differ among species or between males that adopt alternative reproductive phenotypes and/or upon the differential activation of those circuits in different social contexts. However, to date, very little is known about how social stimuli that promote alternative behavioral responses influence AVT circuits within the brain. To address this issue, we exposed adult male goldfish to androstenedione (AD), a pheromonal signal that is released by both males and females during the breeding season, and measured social approach responses of males towards same- and other-sex individuals before and after AD exposure. In a second experiment, we measured AD-induced AVT gene expression using in situ hybridization. We found that brief exposure to AD induces social avoidance in response to rival males, but does not affect the level of sociality exhibited in response to sexually receptive females. Exposure to AD also increases AVT gene expression in the preoptic area of male goldfish, particularly in the parvocellular population of the preoptic nucleus. Together, these data suggest that AD is part of a social signaling system that induces social withdrawal specifically during male-male interactions by activating AVT neurons.

Characterization of the plasticity-related gene, Arc, in the frog brain.

In mammals, expression of the immediate early gene Arc/Arg3.1 in the brain is induced by exposure to novel environments, reception of sensory stimuli, and production of learned behaviors, suggesting a potentially important role in neural and behavioral plasticity. To date, Arc has only been characterized in a few species of mammals and birds, which limits our ability to understand its role in modifying behavior. To begin to address this gap, we identified Arc in two frog species, Xenopus tropicalis and Physalaemus pustulosus, and characterized its expression in the brain of P. pustulosus. We found that the predicted protein for frog Arc shared 60% sequence similarity with Arc in other vertebrates, and we observed high Arc expression in the forebrain, but not the midbrain or hindbrain, of female túngara frogs sacrificed at breeding ponds. We also examined the time‐course of Arc induction in the medial pallium, the homologue of the mammalian hippocampus, in response to a recording of a P. pustulosus mating chorus and found that accumulation of Arc mRNA peaked 0.75 h following stimulus onset. We found that the mating chorus also induced Arc expression in the lateral and ventral pallia and the medial septum, but not in the striatum, hypothalamus, or auditory midbrain. Finally, we examined acoustically induced Arc expression in response to different types of mating calls and found that Arc expression levels in the pallium and septum did not vary with the biological relevance or acoustic complexity of the signal.

Insight into the neuroendocrine basis of signal evolution: a case study in foot-flagging frogs

A hallmark of sexual selection is the evolution of elaborate male sexual signals. Yet, how the physiology of an animal changes to support a new or modified signal is a question that has remained largely unanswered. Androgens are important in regulating male reproductive behavior, therefore, selection for particular signals may drive the evolution of increased androgenic sensitivity in the neuro-motor systems underlying their production. Studies of the neuroendocrine mechanisms of anuran sexual signaling provide evidence to support this idea. Here, we highlight two such cases: first, a large body of work in Xenopus frogs demonstrates that sexually dimorphic androgen receptor (AR) expression in the laryngeal nerves and muscles underlies sexually dimorphic vocal behavior, and second, our own work showing that the recent evolution of a hind limb signal (known as the “foot flag”) in Staurois parvus is accompanied by a dramatic increase in androgenic sensitivity of the thigh muscles that control limb movement. Together, these examples illustrate that the evolutionary modification or gain of a sexual signal is linked with a novel pattern of AR expression in the tissues that support it. We suggest that such co-evolution of AR expression and sex-specific or species-specific signaling behavior exists across vertebrates.