Anna K. Greenwood

Expression of arginine vasotocin in distinct preoptic regions is associated with dominant and subordinate behaviour in an African cichlid fish

Neuropeptides have widespread modulatory effects on behaviour and physiology and are associated with phenotypic transitions in a variety of animals. Arginine vasotocin (AVT) is implicated in mediating alternative male phenotypes in teleost fish, but the direction of the association differs among species, with either higher or lower AVT related to more territorial behaviour in different fishes. To clarify the complex relationship between AVT and alternative phenotype, we evaluated AVT expression in an African cichlid in which social status is associated with divergent behaviour and physiology. We compared AVT mRNA expression between territorial and non-territorial (NT) males in both whole brains and microdissected anterior preoptic areas using transcription profiling, and in individual preoptic nuclei using in situ hybridization. These complementary methods revealed that in the posterior preoptic area (gigantocellular nucleus), territorial males exhibit higher levels of AVT expression than NT males. Conversely, in the anterior preoptic area (parvocellular nucleus), AVT expression is lower in territorial males than NT males. We further correlated AVT expression with behavioural and physiological characteristics of social status to gain insight into the divergent functions of individual AVT nuclei. Overall, our findings highlight a complex association between AVT and social behaviour.

Genetic and Neural Modularity Underlie the Evolution of Schooling Behavior in Threespine Sticklebacks

Although descriptions of striking diversity in animal behavior are plentiful, little is known about the mechanisms by which behaviors change and evolve between groups. To fully understand behavioral evolution, it will be necessary to identify the genetic mechanisms that mediate behavioral change in a natural context. Genetic analysis of behavior can also reveal associations between behavior and morphological or neural phenotypes, providing insight into the proximate mechanisms that control behavior. Relatively few studies to date have successfully identified genes or genomic regions that contribute to behavioral variation among natural populations or species, particularly in vertebrates. Here, we apply genetic approaches to dissect a complex social behavior that has long fascinated biologists, schooling behavior. We performed quantitative trait locus (QTL) analysis of schooling in an F2 intercross between strongly schooling marine and weakly schooling benthic sticklebacks (Gasterosteus aculeatus) and found that distinct genetic modules control different aspects of schooling behavior. Two key components of the behavior, tendency to school and body position when schooling, are uncorrelated in hybrids and map to different genomic regions. Our results further point to a genetic link between one behavioral component, schooling position, and variation in the neurosensory lateral line.

Social Regulation of the Electrical Properties of Gonadotropin-Releasing Hormone Neurons in a Cichlid Fish (Astatotilapia burtoni)

Abstract Variation in reproductive capacity is common across the lives of all animals. In vertebrates, hypothalamic neurons that secrete GnRH are a primary mediator of such reproductive plasticity. Since social interactions suppress gonadal maturity in the African cichlid fish, Astatotilapia (Haplochromis) burtoni, we investigated whether the electrical properties of GnRH neurons were also socially regulated. Adult A. burtoni males are either territorial (T) and reproductively active or nonterritorial (NT) and reproductively regressed, depending upon their social environment. We compared the basic electrical properties of hypothalamic GnRH neurons from T and NT males using whole-cell electrophysiology in vitro. GnRH neurons were spontaneously active and exhibited several different activity patterns. A small fraction of neurons exhibited episodic activity patterns, which have been described in GnRH neurons from mammals. The type of activity pattern and spontaneous firing rate did not vary with reproductive capacity; however, several basic electrical properties were different. Neurons from T males were larger than those from NT males and had higher membrane capacitance and lower input resistance. In neurons from NT males, action potential duration was significantly longer and after-hyperpolarization characteristics were diminished, which led to a tendency for neurons from NT males to fire less rapidly in response to current injection. We predict this could serve to decrease GnRH release in NT males. These data are the first electrophysiological characterization of hypothalamic GnRH neurons in a nonmammalian species and provide evidence for several changes in electrical properties with reproductive state.

Two visual processing pathways are targeted by gonadotropin-releasing hormone in the retina.

In fish the terminal nerve is comprised of a group of cells with somata adjacent to the olfactory bulb and processes that extend both anteriorly to the olfactory mucosa and posteriorly to the telencephalon. In teleost fish an additional group of axons extends along the optic tract and delivers putative neuromodulators to the retina. One peptide – gonadotropin-releasing hormone (GnRH) – has been implicated as a prime candidate neuromodulator based on electrophysiological evidence that exogenous application influences neural activity. Here we describe the expression patterns of two GnRH receptor subtypes in the retina of a teleost fish, Astatotilapia (Haplochromis) burtoni. The type 1 GnRH receptor (GnRH-R1) was expressed in cells of the amacrine cell layer – where lateral inputs affect the flow of visual information from photoreceptors to the brain – and in a distribution and location pattern similar to dopaminergic interplexiform cells. Immunohistochemical labeling of GnRH fibers revealed varicosities along terminal nerve axons near the amacrine cell layer and near cells immunoreactive for tyrosine hydroxylase, a dopaminergic cell marker. This finding supports an existing model that the terminal nerve forms synapses with dopaminergic interplexiform cells. Surprisingly, the type 2 GnRH receptor (GnRH-R2) was abundantly expressed in ganglion cells, which lie along the direct pathway of visual information to the brain. These data suggest that GnRH from the TN could broadly influence processing of retinal signals both in lateral processing circuits through GnRH-R1 and in the vertical throughput pathway through GnRH-R2.

The genetic basis of divergent pigment patterns in juvenile threespine sticklebacks.

Animal pigment patterns are important for a range of functions, including camouflage and communication. Repeating pigment patterns, such as stripes, bars and spots have been of particular interest to developmental and theoretical biologists, but the genetic basis of natural variation in such patterns is largely unexplored. In this study, we identify a difference in a periodic pigment pattern among juvenile threespine sticklebacks (Gasterosteus aculeatus) from different environments. Freshwater sticklebacks exhibit prominent vertical bars that visually break up the body shape, but sticklebacks from marine populations do not. We hypothesize that these distinct pigment patterns are tuned to provide crypsis in different habitats. This phenotypic difference is widespread and appears in most of the freshwater populations that we sampled. We used quantitative trait locus (QTL) mapping in freshwater–marine F2 hybrids to elucidate the genetic architecture underlying divergence in this pigmentation pattern. We identified two QTL that were significantly associated with variation in barring. Interestingly, these QTL were associated with two distinct aspects of the pigment pattern: melanophore number and overall pigment level. We compared the QTL locations with positions of known pigment candidate genes in the stickleback genome. We also identified two major QTL for juvenile body size, providing new insights into the genetic basis of juvenile growth rates in natural populations. In summary, although there is a growing literature describing simple genetic bases for adaptive coloration differences, this study emphasizes that pigment patterns can also possess a more complex genetic architecture.

Differential social regulation of two pituitary gonadotropin-releasing hormone receptors.

In many vertebrates, social interactions regulate reproductive capacity by altering the activity of the hypothalamic-pituitary-gonadal (HPG) axis. To better understand the mechanisms underlying social regulation of reproduction, we investigated the relationship between social status and one main component of the HPG axis: expression levels of gonadotropin-releasing hormone receptor (GnRH-R). Social interactions dictate reproductive capacity in the cichlid fish Astatotilapia burtoni. Reproductively active territory holders suppress the HPG axis of non-territorial males through repeated aggressive encounters. To determine whether the expression of GnRH-R is socially regulated, we quantified mRNA levels of two GnRH-R variants in the pituitaries and brains of territorial (T) and non-territorial (NT) A. burtoni males. We found that T males had significantly higher levels of pituitary GnRH-R1 mRNA than NT males. In contrast, GnRH-R2 mRNA levels in the pituitary did not vary with social status. Pituitaries from both T and NT males expressed significantly higher mRNA levels of GnRH-R1 than GnRH-R2. GnRH mRNA levels in the brain correlated positively with GnRH-R1 mRNA levels in the pituitary but did not correlate with pituitary GnRH-R2. Measurements of GnRH-R1 and GnRH-R2 mRNA levels across the whole brain revealed no social status differences. These results show that, in addition to the known effects of social status on other levels of the HPG axis, GnRH receptor in the pituitary is also a target of social regulation.

Heritable Differences in Schooling Behavior among Threespine Stickleback Populations Revealed by a Novel Assay

Identifying the proximate and ultimate mechanisms of social behavior remains a major goal of behavioral biology. In particular, the complex social interactions mediating schooling behavior have long fascinated biologists, leading to theoretical and empirical investigations that have focused on schooling as a group-level phenomenon. However, methods to examine the behavior of individual fish within a school are needed in order to investigate the mechanisms that underlie both the performance and the evolution of schooling behavior. We have developed a technique to quantify the schooling behavior of an individual in standardized but easily manipulated social circumstances. Using our model school assay, we show that threespine sticklebacks (Gasterosteus aculeatus) from alternative habitats differ in behavior when tested in identical social circumstances. Not only do marine sticklebacks show increased association with the model school relative to freshwater benthic sticklebacks, they also display a greater degree of parallel swimming with the models. Taken together, these data indicate that marine sticklebacks exhibit a stronger tendency to school than benthic sticklebacks. We demonstrate that these population-level differences in schooling tendency are heritable and are shared by individuals within a population even when they have experienced mixed-population housing conditions. Finally, we begin to explore the stimuli that elicit schooling behavior in these populations. Our data suggest that the difference in schooling tendency between marine and benthic sticklebacks is accompanied by differential preferences for social vs. non-social and moving vs. stationary shelter options. Our study thus provides novel insights into the evolution of schooling behavior, as well as a new experimental approach to investigate the genetic and neural mechanisms that underlie this complex social behavior.

Molecular and developmental contributions to divergent pigment patterns in marine and freshwater sticklebacks.

Pigment pattern variation across species or populations offers a tractable framework in which to investigate the evolution of development. Juvenile threespine sticklebacks (Gasterosteus aculeatus) from marine and freshwater environments exhibit divergent pigment patterns that are associated with ecological differences. Juvenile marine sticklebacks have a silvery appearance, whereas sticklebacks from freshwater environments exhibit a pattern of vertical bars. We investigated both the developmental and molecular basis of this population‐level variation in pigment pattern. Time course imaging during the transition from larval to juvenile stages revealed differences between marine and freshwater fish in spatial patterns of chromatophore differentiation as well as in pigment amount and dispersal. In freshwater fish, melanophores appear primarily within dark bars whereas iridophores appear within light bars. By contrast, in marine fish, these chromatophores are interspersed across the flank. In addition to spatially segregated chromatophore differentiation, pigment amount and dispersal within melanophores varies spatially across the flank of freshwater, but not marine fish. To gain insight into the molecular pathways that underlie the differences in pigment pattern development, we evaluated differential gene expression in the flanks of developing fish using high‐throughput cDNA sequencing (RNA‐seq) and quantitative PCR. We identified several genes that were differentially expressed across dark and light bars of freshwater fish, and between freshwater and marine fish. Together, these experiments begin to shed light on the process of pigment pattern evolution in sticklebacks.

Pleiotropic effects of a single gene on skeletal development and sensory system patterning in sticklebacks

BackgroundAdaptation to a new environment can be facilitated by co-inheritance of a suite of phenotypes that are all advantageous in the new habitat. Although experimental evidence demonstrates that multiple phenotypes often map to the same genomic regions, it is challenging to determine whether phenotypes are associated due to pleiotropic effects of a single gene or to multiple tightly linked genes. In the threespine stickleback fish (Gasterosteus aculeatus), multiple phenotypes are associated with a genomic region around the gene Ectodysplasin (Eda), but only the presence of bony lateral plates has been conclusively shown to be caused by Eda.ResultsHere, we ask whether pleiotropy or linkage is responsible for the association between lateral plates and the distribution of the neuromasts of the lateral line. We first identify a strong correlation between plate appearance and changes in the spatial distribution of neuromasts through development. We then use an Eda transgene to induce the formation of ectopic plates in low plated fish, which also results in alterations to neuromast distribution. Our results also show that other loci may modify the effects of Eda on plate formation and neuromast distribution.ConclusionsTogether, these results demonstrate that Eda has pleiotropic effects on at least two phenotypes, highlighting the role of pleiotropy in the genetic basis of adaptation.

Genetic Mapping of Natural Variation in Schooling Tendency in the Threespine Stickleback

Although there is a heritable basis for many animal behaviors, the genetic architecture of behavioral variation in natural populations remains mostly unknown, particularly in vertebrates. We sought to identify the genetic basis for social affiliation in two populations of threespine sticklebacks (Gasterosteus aculeatus) that differ in their propensity to school. Marine sticklebacks from Japan school strongly whereas benthic sticklebacks from a lake in Canada are more solitary. Here, we expanded on our previous efforts to identify quantitative trait loci (QTL) for differences in schooling tendency. We tested fish multiple times in two assays that test different aspects of schooling tendency: 1) the model school assay, which presents fish with a school of eight model sticklebacks; and 2) the choice assay, in which fish are given a choice between the model school and a stationary artificial plant. We found low-to-moderate levels of repeatability, ranging from 0.1 to 0.5, in schooling phenotypes. To identify the genomic regions that contribute to differences in schooling tendency, we used QTL mapping in two types of crosses: benthic × marine backcrosses and an F2 intercross. We found two QTL for time spent with the school in the model school assay, and one QTL for number of approaches to the school in the choice assay. These QTL were on three different linkage groups, not previously linked to behavioral differences in sticklebacks. Our results highlight the importance of using multiple crosses and robust behavioral assays to uncover the genetic basis of behavioral variation in natural populations.