Effective size of a wild salmonid population is greatly reduced by hatchery supplementation

Many declining and commercially important populations are supplemented with captive-born individuals that are intentionally released into the wild. These supplementation programs often create large numbers of offspring from relatively few breeding adults, which can have substantial population-level effects. We examined the genetic effects of supplementation on a wild population of steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, by matching 12 run-years of hatchery steelhead back to their broodstock parents. We show that the effective number of breeders producing the hatchery fish (broodstock parents; N(b)) was quite small (harmonic mean N(b)=25 fish per brood-year vs 373 for wild fish), and was exacerbated by a high variance in broodstock reproductive success among individuals within years. The low N(b) caused hatchery fish to have decreased allelic richness, increased average relatedness, more loci in linkage disequilibrium and substantial levels of genetic drift in comparison with their wild-born counterparts. We also documented a substantial Ryman-Laikre effect whereby the additional hatchery fish doubled the total number of adult fish on the spawning grounds each year, but cut the effective population size of the total population (wild and hatchery fish combined) by nearly two-thirds. We further demonstrate that the Ryman-Laikre effect is most severe in this population when (1) >10% of fish allowed onto spawning grounds are from hatcheries and (2) the hatchery fish have high reproductive success in the wild. These results emphasize the trade-offs that arise when supplementation programs attempt to balance disparate goals (increasing production while maintaining genetic diversity and fitness).     

Structural tuning of the fluorescent protein iLOV for improved photostability

Fluorescent proteins derived from light, oxygen, or voltage (LOV) domains offer advantages over green fluorescent protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein improved LOV (iLOV) was engineered from the blue light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononucleotide (FMN) chromophore; in iLOV, the methyl group of Thr-394 "crowds" the FMN isoalloxazine ring, Leu-470 triggers side chain "flipping" of Leu-472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr-401 and Phe-485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, whereas Ser-390 anchors the side chain of FMN-interacting Gln-489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters.     

Exploring the universe of protein structures beyond the Protein Data Bank

It is currently believed that the atlas of existing protein structures is faithfully represented in the Protein Data Bank. However, whether this atlas covers the full universe of all possible protein structures is still a highly debated issue. By using a sophisticated numerical approach, we performed an exhaustive exploration of the conformational space of a 60 amino acid polypeptide chain described with an accurate all-atom interaction potential. We generated a database of around 30,000 compact folds with at least of secondary structure corresponding to local minima of the potential energy. This ensemble plausibly represents the universe of protein folds of similar length; indeed, all the known folds are represented in the set with good accuracy. However, we discover that the known folds form a rather small subset, which cannot be reproduced by choosing random structures in the database. Rather, natural and possible folds differ by the contact order, on average significantly smaller in the former. This suggests the presence of an evolutionary bias, possibly related to kinetic accessibility, towards structures with shorter loops between contacting residues. Beside their conceptual relevance, the new structures open a range of practical applications such as the development of accurate structure prediction strategies, the optimization of force fields, and the identification and design of novel folds.     

Comparative Analysis of DNA Replication Timing Reveals Conserved Large-Scale Chromosomal Architecture

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome.     

Identification of Novel Inhibitors of Dietary Lipid Absorption Using Zebrafish

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.     

Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry

Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring. Use of selected reaction monitoring is advantageous with complex biological samples and we show this approach can be used to quantify multiple PTMs independently on a single peptide.     

Phonotaxis to male’s calls embedded within a chorus by female gray treefrogs, Hyla versicolor

During the reproductive season, male Hyla versicolor produce advertisement calls to attract females. Females exhibit phonotaxis and approach the individual callers, resulting in amplexus. For frogs that call from dense choruses, the extent to which and the range from which a male’s advertisement call within a chorus can be heard by a receptive female leading to phonotaxis is unclear. We investigated females’ responses to natural choruses in the field and found that they were attracted and showed directed orientation to breeding choruses at distances up to 100 m. To assess the role of acoustic cues in the directed orientation, we conducted acoustic playback experiments in the laboratory using conspecific call and noise as stimuli, as well as chorus sounds (that contained calls from a focal male) recorded at various distances, all played at naturalistic intensities. Using two response metrics (females’ normalized response times and their phonotaxis trajectories) we found that, unlike the field experiments, females oriented and were attracted to chorus sounds from 1 to 32 m only, but not from >32 m, or to band-limited noise. Possible reasons for the observed difference in phonotaxis behavior in the two experimental conditions were discussed.