Brent L. Lockwood

Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening

Larval zebrafish are used extensively for developmental genetic studies due to their salient features, such as small size, external development, optical transparency, and accessibility in large numbers. However, their use for the study of drug and alcohol abuse has not been explored. Here we investigated the response of larval zebrafish to acute treatment of alcohol. Our analyses showed that like adults, the larval zebrafish exhibited a dose-dependent locomotor response to ethanol: intermediate doses led to hyperactivity, whereas high doses have a neurodepressive effect resulting in hypoactivity and sedation. Alcohol also induced morphological changes of melanocytes, providing a visible cellular measure of the biological effects of alcohol in vivo. In addition, alcohol induced thigmotaxis behavior (preference for the edge of a compartment). In the behaviors we analyzed, genetic background influenced the locomotor responses to alcohol. The present study demonstrates that larval zebrafish exert a response to the acute treatment of alcohol, which is genetically modifiable. Therefore, the larval zebrafish represent a tractable vertebrate model system for a large-scale genetic analysis of the biological effects of alcohol.

Transcriptomic responses to heat stress in invasive and native blue mussels (genus Mytilus): molecular correlates of invasive success

SUMMARY Invasive species are increasingly prevalent in marine ecosystems worldwide. Although many studies have examined the ecological effects of invasives, little is known about the physiological mechanisms that might contribute to invasive success. The mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, is a successful invader on the central and southern coasts of California, where it has largely displaced the native congener, Mytilus trossulus. It has been previously shown that thermal responses of several physiological traits may underlie the capacity of M. galloprovincialis to out-compete M. trossulus in warm habitats. To elucidate possible differences in stress-induced gene expression between these congeners, we developed an oligonucleotide microarray with 8874 probes representing 4488 different genes that recognized mRNAs of both species. In acute heat-stress experiments, 1531 of these genes showed temperature-dependent changes in expression that were highly similar in the two congeners. By contrast, 96 genes showed species-specific responses to heat stress, functionally characterized by their involvement in oxidative stress, proteolysis, energy metabolism, ion transport, cell signaling and cytoskeletal reorganization. The gene that showed the biggest difference between the species was the gene for the molecular chaperone small heat shock protein 24, which was highly induced in M. galloprovincialis and showed only a small change in M. trossulus. These different responses to acute heat stress may help to explain – and predict – the invasive success of M. galloprovincialis in a warming world.

Transcriptomic responses to salinity stress in invasive and native blue mussels (genus Mytilus)

The invasive marine mussel Mytilus galloprovincialis has displaced the native congener Mytilus trossulus from central and southern California, but the native species remains dominant at more northerly sites that have high levels of freshwater input. To determine the extent to which interspecific differences in physiological tolerance to low salinity might explain limits to the invasive species’ biogeography, we used an oligonucleotide microarray to compare the transcriptional responses of these two species to an acute decrease in salinity. Among 6777 genes on the microarray, 117 genes showed significant changes that were similar between species, and 12 genes showed significant species‐specific responses to salinity stress. Osmoregulation and cell cycle control were important aspects of the shared transcriptomic response to salinity stress, whereas the genes with species‐specific expression patterns were involved in mRNA splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signalling. Forty‐five genes that changed expression significantly during salinity stress also changed expression during heat stress, but the direction of change in expression was typically opposite for the two forms of stress. These results (i) provide insights into the role of changes in gene expression in establishing physiological tolerance to acute decreases in salinity, and (ii) indicate that transcriptomic differences between M. galloprovincialis and M. trossulus in response to salinity stress are subtle and involve only a minor fraction of the overall suite of gene regulatory responses.

A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish

Transparent larval zebrafish offer the opportunity to unravel genetic and neuronal networks underlying behavior in a developing system. In this study, we developed a choice chamber paradigm to measure reward-associated behavior in larval zebrafish. In the chamber where larval zebrafish have a choice of spending their time in either a water- or morphine-containing compartment, larvae that have previously experienced morphine spend significantly more time in the compartment containing morphine. This behavior can be attentuated by pre-treatment with antagonists of the opioid receptor or the dopamine receptor, and furthermore, is impaired in the too few mutant, which has a genetic deficiency in the production of specific groups of dopaminergic and serotonergic neurons in the ventral forebrain. These results uncover a choice behavior for an addictive substance in larval zebrafish that is mediated through central opioid and monoaminergic neurotransmitter systems.

Ethanol-Modulated Camouflage Response Screen in Zebrafish Uncovers a Novel Role for cAMP and Extracellular Signal-Regulated Kinase Signaling in Behavioral Sensitivity to Ethanol

Ethanol, a widely abused substance, elicits evolutionarily conserved behavioral responses in a concentration-dependent manner in vivo. The molecular mechanisms underlying such behavioral sensitivity to ethanol are poorly understood. While locomotor-based behavioral genetic screening is successful in identifying genes in invertebrate models, such complex behavior-based screening has proven difficult for recovering genes in vertebrates. Here we report a novel and tractable ethanol response in zebrafish. Using this ethanol-modulated camouflage response as a screening assay, we have identified a zebrafish mutant named fantasma (fan), which displays reduced behavioral sensitivity to ethanol. Positional cloning reveals that fan encodes type 5 adenylyl cyclase (AC5). fan/ac5 is required to maintain the phosphorylation of extracellular signal-regulated kinase (ERK) in the forebrain structures, including the telencephalon and hypothalamus. Partial inhibition of phosphorylation of ERK in wild-type zebrafish mimics the reduction in sensitivity to stimulatory effects of ethanol observed in the fan mutant, whereas, strikingly, strong inhibition of phosphorylation of ERK renders a stimulatory dose of ethanol sedating. Since previous studies in Drosophila and mice show a role of cAMP signaling in suppressing behavioral sensitivity to ethanol, our findings reveal a novel, isoform-specific role of AC signaling in promoting ethanol sensitivity, and suggest that the phosphorylation level of the downstream effector ERK is a critical “gatekeeper” of behavioral sensitivity to ethanol.

Maternal loading of a small heat shock protein increases embryo thermal tolerance in Drosophila melanogaster

ABSTRACT Maternal investment is likely to have direct effects on offspring survival. In oviparous animals whose embryos are exposed to the external environment, maternal provisioning of molecular factors like mRNAs and proteins may help embryos cope with sudden changes in the environment. Here, we sought to modify the maternal mRNA contribution to offspring embryos and test for maternal effects on acute thermal tolerance in early embryos of Drosophila melanogaster. We drove in vivo overexpression of a small heat shock protein gene (Hsp23) in female ovaries and measured the effects of acute thermal stress on offspring embryonic survival and larval development. We report that overexpression of the Hsp23 gene in female ovaries produced offspring embryos with increased thermal tolerance. We also found that brief heat stress in the early embryonic stage (0–1 h old) caused decreased larval performance later in life (5–10 days old), as indexed by pupation height. Maternal overexpression of Hsp23 protected embryos against this heat-induced defect in larval performance. Our data demonstrate that transient products of single genes have large and lasting effects on whole-organism environmental tolerance. Further, our results suggest that maternal effects have a profound impact on offspring survival in the context of thermal variability. Highlighted Article: A gene-specific maternal effect confers thermal tolerance to offspring embryos in the fruit fly Drosophila melanogaster.

The environmentally tuned transcriptomes of Mytilus mussels.

ABSTRACT Transcriptomics is a powerful tool for elucidating the molecular mechanisms that underlie the ability of organisms to survive and thrive in dynamic and changing environments. Here, we review the major contributions in this field, and we focus on studies of mussels in the genus Mytilus, which are well-established models for the study of ecological physiology in fluctuating environments. Our review is organized into four main sections. First, we illustrate how the abiotic forces of the intertidal environment drive the rhythmic coupling of gene expression to diel and tidal cycles in Mytilus californianus. Second, we discuss the challenges and pitfalls of conducting transcriptomic studies in field-acclimatized animals. Third, we examine the link between transcriptomic responses to environmental stress and biogeographic distributions in blue mussels, Mytilus trossulus and Mytilus galloprovincialis. Fourth, we present a comparison of transcriptomic datasets and identify 175 genes that share common responses to heat stress across Mytilus species. Taken together, these studies demonstrate that transcriptomics can provide an informative snapshot of the physiological state of an organism within an environmental context. In a comparative framework, transcriptomics can reveal how natural selection has shaped patterns of transcriptional regulation that may ultimately influence biogeography. Summary: Abiotic factors, such as temperature and the tidal cycle, drive patterns of gene expression in Mytilus mussels that underlie whole-organism physiological states, which, in turn, influence biogeographic distributions.

Can the giant snake predict palaeoclimate

Arising from: J. J. Head et al. 457, 715–717 (2009)10.1038/nature07671; Head et al. replyIn their report on Titanoboa cerrejonensis, Head et al. propose that the great size of this 58 to 60 million-year-old snake (estimated length = 13 m, mass = 1,135 kg) indicates a mean annual neotropical temperature (MAT) of 30–34 °C, substantially higher than previous estimates for that period. They argue that the high MAT was necessary to compensate for the decreased mass-specific metabolic rate intrinsic to a snake of this size. However, the relationship on which Head et al. base their conclusion does not account for the scope of behavioural control over body temperature available to Titanoboa due to its huge mass. Our calculations suggest that because of its ability to behaviourally control its body temperature, Titanoboa cannot serve as an accurate palaeothermometer.

Disparate patterns of thermal adaptation between life stages in temperate vs. tropical Drosophila melanogaster

Many terrestrial ectothermic species exhibit limited variation in upper thermal tolerance across latitude. However, these trends may not signify limited adaptive capacity to increase thermal tolerance in the face of climate change. Instead, thermal tolerance may be similar among populations because behavioural thermoregulation by mobile organisms or life stages may buffer natural selection for thermal tolerance. We compared thermal tolerance of adults and embryos among natural populations of Drosophila melanogaster from a broad range of thermal habitats around the globe to assess natural variation of thermal tolerance in mobile vs. immobile life stages. We found no variation among populations in adult thermal tolerance, but embryonic thermal tolerance was higher in tropical strains than in temperate strains. We further report that embryos live closer to their upper thermal limits than adults – that is, thermal safety margins are smaller for embryos than adults. F1 hybrid embryos from crosses between temperate and tropical populations had thermal tolerance that matched that of tropical embryos, suggesting the dominance of heat‐tolerant alleles. Together, our findings suggest that thermal selection has led to divergence in embryonic thermal tolerance but that selection for divergent thermal tolerance may be limited in adults. Further, our results suggest that thermal traits should be measured across life stages to better predict adaptive limits.

Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes

Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result.