Evolutionary conservation of the egr-1 immediate-early gene response in a teleost

Immediate-early gene expression is a key part of a neuron's response to behaviorally relevant stimuli and, as a result, localization of immediate-early gene expression can be a useful marker for neural activity. We characterized the immediate-early gene egr-1 (also called zif268, NGFI-A, krox-24, ZENK) in the teleost Astatotilapia (Haplochromis) burtoni. We compared the A. burtoni egr-1 predicted protein sequence to that of other vertebrates, characterized its gene expression time course, and localized its induced expression throughout the brain. The A. burtoni egr-1 predicted protein shared putative functional domains with egr-1 of other vertebrates and shared 81% sequence similarity with zebrafish and 66% with mouse. We identified distinct mammalian and teleost inserts rich in serine residues within one activation domain, suggesting convergent responses to selection pressures to increase the number of serine residues in this region. Functionally, we found that A. burtoni egr-1 gene expression peaked near 30 minutes after pharmacological stimulation and thereby displayed the transient expression above basal levels characteristic of egr-1 expression in birds and mammals. Finally, we observed distinct patterns of egr-1 gene induction in the brain by natural and pharmacological stimuli. Unstimulated males had very low expression levels of egr-1, whereas males stimulated by their normal environment showed higher levels of expression specific to particular brain regions. Males injected with a glutamate receptor agonist also had region-specific induction of egr-1 expression. We conclude that the egr-1 immediate-early gene response is evolutionarily conserved and will, therefore, be useful for identifying functional neural responses in nontraditional model species.     

Electrical synapses connect a network of gonadotropin releasing hormone neurons in a cichlid fish

Initiating and regulating vertebrate reproduction requires pulsatile release of gonadotropin-releasing hormone (GnRH1) from the hypothalamus. Coordinated GnRH1 release, not simply elevated absolute levels, effects the release of pituitary gonadotropins that drive steroid production in the gonads. However, the mechanisms underlying synchronization of GnRH1 neurons are unknown. Control of synchronicity by gap junctions between GnRH1 neurons has been proposed but not previously found. We recorded simultaneously from pairs of transgenically labeled GnRH1 neurons in adult male Astatotilapia burtoni cichlid fish. We report that GnRH1 neurons are strongly and uniformly interconnected by electrical synapses that can drive spiking in connected cells and can be reversibly blocked by meclofenamic acid. Our results suggest that electrical synapses could promote coordinated spike firing in a cellular assemblage of GnRH1 neurons to produce the pulsatile output necessary for activation of the pituitary and reproduction.Development and function of the reproductive system in vertebrates depends on the timing and levels of signaling by gonadal sex steroids (1, 2). Production of these steroids is controlled by neurons expressing gonadotropin-releasing hormone (GnRH1), which comprise the final output of the brain to the hypothalamic-pituitary-gonadal axis. During vertebrate development, GnRH1 neurons originate outside the central nervous system in the olfactory placode and migrate into the basal forebrain (3–6). These neurons signal to the pituitary via the decapeptide GnRH1 to effect the release of the gonadotropins, follicle stimulating hormone and luteinizing hormone, which in turn stimulate steroid production by the gonads. It has long been known that this release depends on coordinated, pulsatile GnRH1 release, not simply elevated levels (7, 8), requiring some level of synchronization in the output of these neurons. Episodic activation of the pituitary gonadotropes has been observed in multiple vertebrate taxa, including mammals and fish (9–12), however, mechanisms that underlie this required coordinated activity of GnRH1 neurons are unknown. Synchrony could in principle derive from coincident input from a “pacemaker” neural population, from direct coupling of GnRH1 neurons, or from a combination of mechanisms. Gap junction-mediated coupling has been suspected to play a role, as synchronous firing can be observed in neurons mechanically isolated from brain slices and in cultures of embryonic mouse and primate neurons, and immortalized mouse GnRH1 neurons express the connexin proteins that constitute gap junctions (13–15). However, no evidence for gap junctions among adult GnRH1 cells in vivo has been found (16, 17).To search for the origin of synchrony among these neurons, we used a unique model system for analysis of GnRH1 neurons, Astatotilapia burtoni, a cichlid fish. GnRH1 neurons in males of this species exhibit dynamic morphological plasticity caused by changes in their social status (18–21). Here we use transgenic dominant male A. burtoni to perform paired recordings from GnRH1 neurons, and report that they are reciprocally connected by electrical synapses. These findings suggest that gap junctions contribute to the coordinated firing of these neurons necessary for reproductive function.     

Ontogenetic divergence generates novel phenotypes in hybrid cichlids

Hybridization is suggested to contribute to ecomorphological and taxonomic diversity in lacustrine East African cichlids. This is supported by studies demonstrating that genetic diversity within lake radiations has been influenced by hybridization events, leading to extensive phenotypic differentiation of genetically closely related species. Hybrid persistence and speciation in sympatry with gene flow can be explained by pleiotropy in traits involved in reproductive isolation; however, little attention has been given to how trait differentiation is established during hybrid ontogeny, and how this may relate to trophic and locomotor specialization. This study compares body shape changes in a Lake Victoria cichlid hybrid throughout its post-hatch ontogeny to those of its parental species. Across the considered age/size categories, hybrids occupy a distinct and intermediate morphological space, yet where several transgressive traits emerge. A between-group principal component analysis on body shapes across size categories reveals axes of shape variation exclusive to the hybrids in the youngest/smallest size categories. Shape differences in the hybrids involved morphological traits known to be implicated in trophic and locomotor specializations in the parental species. Combined, our findings suggest that phenotypic divergence in the hybrid can lead to functional differences that may potentially release them to some degree from competition with the parental species. These findings agree with recent literature that addresses the potential importance of hybridization for the unusually recent origin of the Lake Victoria cichlid super-species flock.     

Ecological consequences of early Late Pleistocene megadroughts in tropical Africa

Extremely arid conditions in tropical Africa occurred in several discrete episodes between 135 and 90 ka, as demonstrated by lake core and seismic records from multiple basins [Scholz CA, Johnson TC, Cohen AS, King JW, Peck J, Overpeck JT, Talbot MR, Brown ET, Kalindekafe L, Amoako PYO, et al. (2007) Proc Natl Acad Sci USA 104:16416-16421]. This resulted in extraordinarily low lake levels, even in Africa's deepest lakes. On the basis of well dated paleoecological records from Lake Malawi, which reflect both local and regional conditions, we show that this aridity had severe consequences for terrestrial and aquatic ecosystems. During the most arid phase, there was extremely low pollen production and limited charred-particle deposition, indicating insufficient vegetation to maintain substantial fires, and the Lake Malawi watershed experienced cool, semidesert conditions (<400 mm/yr precipitation). Fossil and sedimentological data show that Lake Malawi itself, currently 706 m deep, was reduced to an approximately 125 m deep saline, alkaline, well mixed lake. This episode of aridity was far more extreme than any experienced in the Afrotropics during the Last Glacial Maximum (approximately 35-15 ka). Aridity diminished after 95 ka, lake levels rose erratically, and salinity/alkalinity declined, reaching near-modern conditions after 60 ka. This record of lake levels and changing limnological conditions provides a framework for interpreting the evolution of the Lake Malawi fish and invertebrate species flocks. Moreover, this record, coupled with other regional records of early Late Pleistocene aridity, places new constraints on models of Afrotropical biogeographic refugia and early modern human population expansion into and out of tropical Africa.     

Effects of alcohol,congeners, and acetaldehyde on aggressive behavior of the convict cichlid

Ethanol and three beverage alcohols were examined for their effects on the aggressive responses of the convict cichlid. At an ethanol concentration that gave negligible effects on the basal level of these responses, rum was observed to depress aggression. As its concentration of acetaldehyde was significantly higher than that of the other beverage alcohols, we assessed the effects of acetyldehyde on the aggressive responses, as well as the locomotor activity of the fish. At 4mg/l, acetaldehyde completely inhibited aggressive responses, without affecting locomotor activity. It is likely, however, that acetaldehyde does not explain the entire inhibitory effect of rum on the aggressive responses of the cichlid.     

Distribution of LPXRFa,a gonadotropin‐inhibitory hormone ortholog peptide,and LPXRFa receptor in the brain and pituitary of the tilapia

In vertebrates, gonadotropin‐releasing hormone (GnRH) and gonadotropin‐inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa‐R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa‐R has not been studied in teleost species, partially because of the lack of fish‐specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa‐immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa‐R‐immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa‐immunoreactive fibers nor LPXRFa‐R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle‐stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α‐melanotropin (α‐MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa‐R are expressed only in LH, ACTH, and α‐MSH cells. These results suggest that LPXRFa and LPXRFa‐R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753–2775, 2016. © 2016 Wiley Periodicals, Inc.     

Continuous 1.3-million-year record of East African hydroclimate,and implications for patterns of evolution and biodiversity

The transport of moisture in the tropics is a critical process for the global energy budget and on geologic timescales, has markedly influenced continental landscapes, migratory pathways, and biological evolution. Here we present a continuous, first-of-its-kind 1.3-My record of continental hydroclimate and lake-level variability derived from drill core data from Lake Malawi, East Africa (9–15° S). Over the Quaternary, we observe dramatic shifts in effective moisture, resulting in large-scale changes in one of the world’s largest lakes and most diverse freshwater ecosystems. Results show evidence for 24 lake level drops of more than 200 m during the Late Quaternary, including 15 lowstands when water levels were more than 400 m lower than modern. A dramatic shift is observed at the Mid-Pleistocene Transition (MPT), consistent with far-field climate forcing, which separates vastly different hydroclimate regimes before and after ∼800,000 years ago. Before 800 ka, lake levels were lower, indicating a climate drier than today, and water levels changed frequently. Following the MPT high-amplitude lake level variations dominate the record. From 800 to 100 ka, a deep, often overfilled lake occupied the basin, indicating a wetter climate, but these highstands were interrupted by prolonged intervals of extreme drought. Periods of high lake level are observed during times of high eccentricity. The extreme hydroclimate variability exerted a profound influence on the Lake Malawi endemic cichlid fish species flock; the geographically extensive habitat reconfiguration provided novel ecological opportunities, enabling new populations to differentiate rapidly to distinct species.Insolation forcing of tropical convection and shifts in the Intertropical Convergence Zone (ITCZ) are considered principal mechanisms driving tropical climate variability on geologic time scales (1–5). However, instrumental records show that the ITCZ over land is poorly characterized compared with the oceans (Fig. 1), and the transport of oceanic moisture to the hinterlands is complex, because topographic barriers redirect winds and block advection to the continental interiors (6). To document moisture transport onto the continents over geological time scales, terrestrial records of tropical paleoclimate with the length and continuity of ocean drilling records are required. Continental drilling in long-lived tropical lakes provides continuous, high-resolution paleoclimate records that extend well past the last glacial maximum (7, 8), and augment shorter, well-dated records from outcrops. Lakes of great antiquity also contain numerous endemic species—notably, cichlid fishes—whose modern assemblages evolved in concert with changing climates, and which figure prominently into models of speciation and diversification (9, 10).Open in a separate windowFig. 1.(A and B) Continental TRMM 2B31 [combined TRMM microwave imager (TMI) and precipitation radar (PR)] precipitation estimates from 1998 to 2009 for January and July (SI Appendix) across Africa. Drill site 1 from the Lake Malawi Drilling Project and the interpreted ITCZ and CAB are labeled. (C) Bathymetric map of Lake Malawi (maximum water depths of north and central basin are ∼600 and ∼700 m, respectively), with digital elevation model of the region surrounding the lake. The lake surface elevation is 474 m, and maximum relief on map is ∼2,600 m. Red circle is central basin drill site. Yellow circle is location of Rungwe volcanoes, the presumptive source of the tephras dated in the core. Main border faults, which accommodate most of the basin subsidence, are shown in red.     

Two new species of <Emphasis Type="Italic">Sciadicleithrum</Emphasis> (Monogenea,Dactylogyridae) parasites of Neotropical cichlid fishes from the Paraná River,Brazil

Two new species of Sciadicleithrum are described from the gills of Neotropical cichlid fishes collected from Paraná River, Brazil. Sciadicleithrum satanopercae sp. nov. is described from the gills of Satanoperca pappaterra and differs from congeners by having a dorsal anchor with the distal portion complex and the inner margin with a “denticulum” between shaft and point. Sciadicleithrum joanae sp. nov. is described from the gills of Crenicichla niederleinii and C. britskii collected from Paraná River. Sciadicleithrum joanae sp. nov. differs from congeners by having a male copulatory organ comprising about half a clockwise loop and a vaginal pore “bulb-shaped”.     

From the Cover: Brain diversity evolves via differences in patterning

Differences in brain region size among species are thought to arise late in development via adaptive control over neurogenesis, as cells of previously patterned compartments proliferate, die, and/or differentiate into neurons. Here we investigate comparative brain development in ecologically distinct cichlid fishes from Lake Malawi and demonstrate that brains vary among recently evolved lineages because of early patterning. Divergence among rock-dwellers and sand-dwellers in the relative size of the telencephalon versus the thalamus is correlated with gene expression variation in a regulatory circuit (composed of six3, fezf2, shh, irx1b, and wnt1) known from model organisms to specify anterior-posterior (AP) brain polarity and position the shh-positive signaling boundary zona limitans intrathalamica (ZLI) in the forebrain. To confirm that changes in this coexpression network are sufficient to produce the differences we observe, we manipulated WNT signaling in vivo by treating rock-dwelling cichlid embryos with temporally precise doses of LiCl. Chemically treated rock-dwellers develop gene expression patterns, ZLIs, and forebrains distinct from controls and untreated conspecifics, but strongly resembling those of sand-dwellers. Notably, endemic Malawi rock- and sand-dwelling lineages are alternately fixed for an SNP in irx1b, a mediator of WNT signaling required for proper thalamus and ZLI. Together, these natural experiments in neuroanatomy, development, and genomics suggest that evolutionary changes in AP patterning establish ecologically relevant differences in the elaboration of cichlid forebrain compartments. In general, variation in developmental patterning might lay the foundations on which neurogenesis erects diverse brain architectures.