BackgroundNext-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history.ResultsWe generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome.ConclusionsWe show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression.

A major goal of modern evolutionary biology is to understand the causes and consequences of phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes in response to variable environments. While ecological and quantitative genetic studies have evaluated models of the evolution of adaptive plasticity, some long-standing questions about plasticity require more mechanistic approaches. Here, we address two of those questions: does plasticity facilitate adaptive evolution? And do physiological costs place limits on plasticity? We examine these questions by comparing genetically and plastically regulated behavioural variation in sailfin mollies (Poecilia latipinna), which exhibit striking variation in plasticity for male mating behaviour. In this species, some genotypes respond plastically to a change in the social environment by switching between primarily courting and primarily sneaking behaviour. In contrast, other genotypes have fixed mating strategies (either courting or sneaking) and do not display plasticity. We found that genetic and plastic variation in behaviour were accompanied by partially, but not completely overlapping changes in brain gene expression, in partial support of models that predict that plasticity can facilitate adaptive evolution. We also found that behavioural plasticity was accompanied by broader and more robust changes in brain gene expression, suggesting a substantial physiological cost to plasticity. We also observed that sneaking behaviour, but not courting, was associated with upregulation of genes involved in learning and memory, suggesting that sneaking is more cognitively demanding than courtship.

Some human subjects report vestibular disturbances such as vertigo, apparent motion, and nausea around or within high strength MRI systems operating at 4 T to 9.4 T. These vestibular effects have been ascribed to the consequences of movement through the high magnetic field. We have previously found that exposure to magnetic fields above 7 T suppresses rearing, causes locomotor circling, and induces conditioned taste aversion (CTA) in rodents. The present experiments were designed to test the effects on rats of motion through the magnetic field of the 14.1 T superconducting magnet. In experiment 1, we compared the effects of multiple rapid insertions and removals from the center of the magnet to the effects of continuous exposure. Repeated traversal of the magnetic field gradient with only momentary exposure to 14.1 T was sufficient to suppress rearing and induce a significant CTA. Repeated insertion and removal from the magnet, however, did not have a greater effect than a single 30-min exposure on either acute locomotor behavior or CTA acquisition. Prolonged exposure was required to induce locomotor circling. In the second series of experiments, we controlled the rate of insertion and removal by means of an electric motor. Locomotor circling appeared to be dependent on the speed of insertion and removal, but the suppression of rearing and the acquisition of CTA were independent of speed of insertion and removal. In experiment 3, we inserted rats into the center of the magnet and then rotated them about their rostral-caudal axis during a 30-min 14.1 T exposure. Rotation within the magnet did not modulate the behavioral effects of exposure. We conclude that, in rats, movement through the steep gradient of a high magnetic field has some behavioral effects, but sustained exposure to the homogenous center of the field is required for the full behavioral consequences.

Exposure of rats to high strength static magnetic fields of 7 T or above has behavioral effects such as the induction of locomotor circling, the suppression of rearing, and the acquisition of conditioned taste aversion (CTA). To determine if habituation occurs across magnetic field exposures, rats were pre-exposed two times to a 14 T static magnetic field for 30 min on two consecutive days; on the third day, rats were given access to a novel 0.125% saccharin prior to a third 30-min exposure to the 14 T magnetic field. Compared to sham-exposed rats, pre-exposed rats showed less locomotor circling and an attenuated CTA. Rearing was suppressed in all magnet-exposed groups regardless of pre-exposure, suggesting that the suppression of rearing is more sensitive than other behavioral responses to magnet exposure. Habituation was also observed when rats under went pre-exposures at 2-3 hour intervals on a single day. Components of the habituation were also long lasting; a diminished circling response was observed when rats were exposed to magnetic field 36 days after 2 pre-exposures. To control for possible effects of unconditioned stimulus pre-exposure, rats were also tested in a similar experimental design with two injections of LiCl prior to the pairing of saccharin with a third injection of LiCl. Pre-exposure to LiCl did not attenuate the LiCl-induced CTA, suggesting that 2 pre-exposures to an unconditioned stimulus are not sufficient to explain the habituation to magnet exposure. Because the effects of magnetic field exposure are dependent on an intact vestibular apparatus, and because the vestibular system can habituate to many forms of perturbation, habituation to magnetic field exposure is consistent with mediation of magnetic field effects by the vestibular system.