Neural activation patterns associated with mouthbrooding, maternal care, infanticide and fry release in an African cichlid fish.

Parental care has evolved several times and is present across taxa. Parental care behaviors, such as food provisioning and protection, are critical for offspring success. However, infanticide can co-exist with parental care in the same species. The mechanisms underlying the switch from care to consumption and from offspring dependence to independence are relatively unknown, especially in fishes, the oldest and largest group of vertebrates. Mouthbrooding, an extreme example of parental care present in dozens of genera of fishes, provides an excellent opportunity to investigate the brain regions important for parental care. The maternal mouthbrooding African cichlid fish Astatotilapia burtoni broods developing young inside the mouth for approximately 14 days, then provides post-release maternal care by protecting fry inside the mouth when threatened. Following the post-release maternal care phase, females can exhibit infanticide and consume their own offspring. We used immunohistochemistry for the neural activation marker pS6 to identify differences in neural activation among mouthbrooding, maternal-care-providing and infanticide-exhibiting females, and between pre- and post-release fry. We identified five brain regions (Dc-5, ATn, nPPa, Vd-c and Dl-g) that are differentially activated among mouthbrooding, maternal care and infanticide females as well as six regions (Dm, Vv, Vd, Vs-m, TPp, PGZ and INL of retina) differentially activated between pre- and post-release fry. This study identifies both shared and distinct circuitry that may support transitions between parental care states and from care to infanticide, as well as regions in developed fry that support the transition from pre- to post-release.

Galanin neuron activation in feeding, parental care, and infanticide in a mouthbrooding African cichlid fish.

Galanin is a conserved neuropeptide involved in parental care and feeding. While galanin is known to mediate parental care and infanticide in rodents, its role in parental care and feeding behaviors in other taxa, particularly fishes, remains poorly understood. Mouthbrooding is an extreme form of parental care common in fishes in which caregivers carry offspring in their buccal cavity for the duration of development, resulting in obligatory starvation. In the cichlid fish Astatotilapia burtoni, females brood their young for ~2 wks and perform maternal care after release by collecting them into their mouth when threatened. However, females will cannibalize their brood after ~5 days. To examine the role of gal in feeding and maternal care, we collected mouthbrooding, fed, and starved females, as well as those displaying post-release maternal care and infanticide behaviors. Activation of gal neurons in the preoptic area (POA) was associated with parental care, providing the first link between gal and offspring-promoting behaviors in fishes. In contrast, activation of gal neurons in the lateral tuberal nucleus (NLT), the Arcuate homolog, was associated with feeding and infanticide. Overall, these data suggest gal is functionally conserved across vertebrate taxa with POA gal neurons promoting maternal care and Arc/NLT gal neurons promoting feeding.

Context-dependent chemosensory signaling, aggression and neural activation patterns in gravid female African cichlid fish.

Social animals must constantly assess their environment to make appropriate behavioral decisions. The use of various sensory modalities is imperative in this process and it is hypothesized that the highly conserved brain nuclei comprising the social decision-making network (SDMN) integrates social information with an animal's internal state to elicit behavioral responses. Here, we used the highly social African cichlid fish, Astatotilapia burtoni, to investigate whether reproductively receptive (gravid) females show contextual chemosensory signaling, social behaviors and neural activation patterns within the SDMN. We exposed gravid females to different social contexts: (1) dominant male (inter-sexual reproductive); (2) mouth brooding (non-receptive) female; (3) gravid female (intra-sexual aggressive); (4) juvenile fish (low social salience); and (5) empty compartment (control). By injecting females with a blue dye to visualize urine pulses, we found that gravid females show context-dependent urination, exhibiting higher urination rates in the presence of dominant males (reproductive context) and mouth brooding females (aggressive contexts). Further, gravid females show contextual aggression with increased aggressive displays toward mouth brooding females compared with other gravid females. Using in situ hybridization to quantify cells expressing the immediate early gene cfos as a measure of neural activation, we also show that certain regions of the SDMN in gravid females are differentially activated after exposure to high compared with low social salience contexts. Coupled with previous reports, these results demonstrate true chemosensory communication in both sexes of a single fish species, as well as reveal the neural substrates mediating intra- and inter-sexual social behaviors in females.

Reproductive and metabolic state differences in olfactory responses to amino acids in a mouth brooding African cichlid fish.

Olfaction mediates many crucial life-history behaviors such as prey detection, predator avoidance, migration and reproduction. Olfactory function can also be modulated by an animal's internal physiological and metabolic states. While this is relatively well studied in mammals, little is known about how internal state impacts olfaction in fishes, the largest and most diverse group of vertebrates. Here we apply electro-olfactograms (EOGs) in the African cichlid fish Astatotilapia burtoni to test the hypothesis that olfactory responses to food-related cues (i.e. l-amino acids; alanine and arginine) vary with metabolic, social and reproductive state. Dominant males (reproductively active, reduced feeding) had greater EOG magnitudes in response to amino acids at the same tested concentration than subordinate males (reproductively suppressed, greater feeding and growth rates). Mouth brooding females, which are in a period of starvation while they brood fry in their mouths, had greater EOG magnitudes in response to amino acids at the same tested concentration than both recovering and gravid females that are feeding. Discriminant function analysis on EOG magnitudes also grouped the male (subordinate) and female (recovering, gravid) phenotypes with higher food intake together and distinguished them from brooding females and dominant males. The slope of the initial negative phase of the EOG also showed intra-sexual differences in both sexes. Our results demonstrate that the relationship between olfaction and metabolic state observed in other taxa is conserved to fishes. For the first time, we provide evidence for intra-sexual plasticity in the olfactory response to amino acids that is influenced by fish reproductive, social and metabolic state.

Swim bladder morphology changes with female reproductive state in the mouth-brooding African cichlid Astatotilapia burtoni.

Mouth brooding is an extreme form of parental care in which the brooding parent carries the developing young in their buccal cavity for the duration of development. Brooding fish need to compensate for the brood weight on the anterior portion of their body. For fishes with a compartmentalized swim bladder, gas distribution between the chambers may aid in regulating buoyancy during brooding. To test this hypothesis, we took radiographs of Astatotilapia burtoni to compare the swim bladder morphology of gravid, mouth-brooding and recovering females. Following spawning, females carry developing fish in their buccal cavity for ∼2 weeks, resulting in a larger and rounder anterior swim bladder compartment. Comparatively, the swim bladder of gravid females is long and cylindrical. Using small beads to mimic brood weight and its effects on female buoyancy, swim bladder changes were induced that resembled those observed during brooding. Immediately after releasing their fry, brooding females swim at a positive angle of attack but correct their swimming posture to normal within 5 min, suggesting a rapid change in swim bladder gas distribution. These data provide new insights into how swim bladder morphology and swimming behavior change during mouth brooding, and suggest a compartmentalized swim bladder may be a morphological adaptation for mouth brooding.

Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish.

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parent's mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation.

Contextual chemosensory urine signaling in an African cichlid fish.

Chemosensory signaling is crucial for communication in many fish species, but little is known about how signalers modulate chemical output in response to sensory information and social context. Here, we tested the hypothesis that dominant male African cichlid fish (Astatotilapia burtoni) use urine signals during social interactions, and demonstrate that this signaling depends on social context (reproductive; territorial) and on available sensory information (visual cues; full interaction). We injected males with dye to visualize urine pulses and exposed them to full sensory information or visual cues alone of four types: (1) dominant male; (2) gravid (reproductively receptive) females; (3) mouth-brooding (non-receptive) females; or (4) control (no fish). We found that males released urine sooner and increased their urination frequency when visually exposed to gravid females as compared with mouth-brooding females and or no-fish controls. While males could distinguish female reproductive states using visual cues alone, courtship behavior rates were ∼10-fold higher when they fully interacted with gravid females compared with receiving visual cues alone. Males also increased their urination and territorial behaviors when exposed to another male, suggesting that chemical signals may convey information on dominance status. These data support the hypothesis that dominant males use urine as a chemical signal and adjust the frequency of their urine output based on contextual information.

Noise during mouthbrooding impairs maternal care behaviors and juvenile development and alters brain transcriptomes in the African cichlid fish Astatotilapia burtoni.

Anthropogenic noise has increased underwater ambient sound levels in the range in which most fishes detect and produce acoustic signals. Although the impacts of increased background noise on fish development have been studied in a variety of species, there is a paucity of information on how noise affects parental care. Mouthbrooding is an energetically costly form of parental care in which the brooding fish carries developing larvae in the buccal cavity for the duration of development. In the African cichlid Astatotilapia burtoni, females carry their brood for ~2 weeks during which time they do not eat. To test the hypothesis that increased background noise impacts maternal care behaviors and brood development, we exposed brooding females to a 3-h period of excess noise (~140 dB) played through an underwater speaker. Over half of noise-exposed brooding females cannibalized or pre-maturely released their brood, but 90% of control females exhibited normal brooding behaviors. RNA-seq analysis revealed that transcripts related to feeding and parental care were differentially expressed in the brains of noise-exposed females. Juveniles that were exposed to noise during their brood period within the mother's mouth had lower body condition factors, higher mortality and altered head transcriptomes compared with control broods. Furthermore, onset of adult-typical coloration and behaviors was delayed compared with control fish. Together, these data indicate that noise has severe impacts on reproductive fitness in mouthbrooding females. Our results, combined with past studies, indicate that parental care stages are extremely susceptible to noise-induced perturbations with detrimental effects on species persistence.

Distribution of aromatase in the brain of the African cichlid fish Astatotilapia burtoni: Aromatase expression, but not estrogen receptors, varies with female reproductive-state.

Estrogen synthesis and signaling in the brains of vertebrates has pleotropic effects ranging from neurogenesis to modulation of behaviors. The majority of studies on brain-derived estrogens focus on males, but estrogenic signaling in females likely plays important roles in regulation of reproductive cycling and social behaviors. We used females of the mouth brooding African cichlid fish, Astatotilapia burtoni, to test for reproductive state-dependent changes in estrogenic signaling capacity within microdissected brain nuclei that are important for social behaviors. Expression levels of the rate-limiting enzyme aromatase, but not estrogen receptors, measured by qPCR changes across the reproductive cycle. Gravid females that are close to spawning had higher aromatase levels in all brain regions compared to females with lower reproductive potential. This brain aromatase expression was positively correlated with circulating estradiol levels and ovarian readiness. Using chromogenic in situ hybridization we localized aromatase-expressing cells to ependymal regions bordering the ventricles from the forebrain to the hindbrain, and observed more abundant staining in gravid compared to mouth brooding females in most regions. Staining was most prominent in subpallial telencephalic regions, and diencephalic regions of the preoptic area, thalamus, and hypothalamus, but was also observed in sensory and sensorimotor areas of the midbrain and hindbrain. Aromatase expression was observed in radial glial cells, revealed by co-localization with the glial marker GFAP and absence of co-localization with the neuronal marker HuC/D. Collectively these results support the idea that brain-derived estradiol in females may serve important functions in reproductive state-dependent physiological and behavioral processes across vertebrates.

Distribution and female reproductive state differences in orexigenic and anorexigenic neurons in the brain of the mouth brooding African cichlid fish, Astatotilapia burtoni.

Integration of reproduction and metabolism is necessary for species survival. While the neural circuits controlling energy homeostasis are well-characterized, the signals controlling the relay of nutritional information to the reproductive axis are less understood. The cichlid fish Astatotilapia burtoni is ideal for studying the neural regulation of feeding and reproduction because females cycle between a feeding gravid state and a period of forced starvation while they brood developing young inside their mouths. To test the hypothesis that candidate neuropeptide-containing neurons known to be involved in feeding and energy homeostasis in mammals show conserved distribution patterns, we performed immunohistochemistry and in situ hybridization to localize appetite-stimulating (neuropeptide Y, NPY; agouti-related protein, AGRP) and appetite-inhibiting (cocaine and amphetamine-regulated transcript, CART; pro-opiomelanocortin, pomc1a) neurons in the brain. NPY, AGRP, CART, and pomc1a somata showed distribution patterns similar to other teleosts, which included localization to the lateral tuberal nucleus (NLT), the putative homolog of the mammalian arcuate nucleus. Gravid females also had larger NPY and AGRP neurons in the NLT compared to brooding females, but brooding females had larger pomc1a neurons compared to gravid females. Hypothalamic agrp mRNA levels were also higher in gravid compared to brooding females. Thus, larger appetite-stimulating neurons (NPY, AGRP) likely promote feeding while females are gravid, while larger pomc1a neurons may act as a signal to inhibit food intake during mouth brooding. Collectively, our data suggest a potential role for NPY, AGRP, POMC, and CART in regulating energetic status in A. burtoni females during varying metabolic and reproductive demands.

Morphology of the mechanosensory lateral line system in the Atlantic Stingray, Dasyatissabina: The mechanotactile hypothesis.

The biological function of anatomical specializations in the mechanosensory lateral line of elasmobranch fishes is essentially unknown. The gross and histological features of the lateral line in the Atlantic stingray, Dasyatis sabina, were examined with special reference to its role in the localization and capture of natural invertebrate prey. Superficial neuromasts are arranged in bilateral rows near the dorsal midline from the spiracle to the posterior body disk and in a lateral position along the entire length of the tail. All dorsal lateral line canals are pored, contain sensory neuromasts, and have accessory lateral tubules that most likely function to increase their receptive field. The pored ventral canal system consists of the lateral hyomandibular canal along the disk margin and the short, separate mandibular canal on the lower jaw. The extensive nonpored and relatively compliant ventral infraorbital, supraorbital, and medial hyomandibular canals form a continuous complex on the snout, around the mouth, and along the abdomen. Vesicles of Savi are small mechanosensory subdermal pouches that occur in bilateral rows only along the ventral midline of the rostrum. Superficial neuromasts are best positioned to detect water movements along the transverse body axis such as those produced by tidal currents, conspecifics, or predators. The pored dorsal canal system is positioned to detect water movements created by conspecifics, predators, or possibly distortions in the flow field during swimming. Based upon the stingray lateral line morphology and feeding behavior, we propose the Mechanotactile Hypothesis, which states that the ventral nonpored canals and vesicles of Savi function as specialized tactile mechanoreceptors that facilitate the detection and capture of small benthic invertebrate prey. J. Morphol. 238:1-22, 1998. © 1998 Wiley-Liss, Inc.

Test of the mechanotactile hypothesis: neuromast morphology and response dynamics of mechanosensory lateral line primary afferents in the stingray.

The mechanotactile hypothesis proposes that ventral non-pored lateral line canals in the stingray function to facilitate localization of prey that contact the skin during benthic feeding. This study used comparative neurophysiological and morphological techniques to test whether ventral non-pored canals encode the velocity of skin movements, and show other adaptations that may enhance detection of tactile stimuli from their prey. Resting discharge rate of lateral line primary afferent neurons was lower among units from ventral than dorsal canal groups. The ventral non-pored canals had a higher proportion of silent units (31%) than either ventral (3%) or dorsal (13%) pored canals, thus may have an enhanced potential for detection of phasic contact with prey. Primary afferents from pored canals showed response characteristics consistent with acceleration detectors, with best frequencies of 20-30 Hz. In contrast, units from non-pored canals responded to tactile skin depression velocities of 30-630 microm s(-1) from 1-20 Hz, and encoded the velocity of canal fluid induced by skin movement with best frequencies of < or =10 Hz. Sensitivity of non-pored canals to direct skin depression velocity was 2-10 times greater than to hydrodynamic dipole stimulation near the skin. No morphological specialization of hair cell orientation was found among pored and non-pored canals. These low frequency, tactile response properties support the hypothesis that the stingray's non-pored ventral lateral line functions as a mechanotactile receptor subsystem used to guide small benthic invertebrates to the ventrally positioned mouth.