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bioRxiv2014

Behavioral variation in Drosophila melanogaster: no evidence for common alleles of large- effect at the foraging gene in a population from North Carolina, USA

Thirty four years ago, it was postulated that natural populations of Drosophila melanogaster are comprised of two behavioral morphs termed \"rover\" and \"sitter\", and that this variation is caused mainly by large-effect alleles at a single locus. Since that time, considerable data has been amassed that compares the behavior and physiology of these morphs. Contrary to common assertions, however, published support for the existence of common large effect alleles in nature is quite limited. To further investigate, we quantified the foraging behavior of 36 natural strains, performed a genome-wide association study, and described patterns of molecular evolution at the foraging locus. Though there was significant variation in foraging behavior among genotypes, this variation was continuously distributed and not significantly associated with genetic variation at the foraging gene. Patterns of molecular population genetic variation at this gene also provide no support for the hypothesis that for is a target of long term balancing selection We propose that additional data is required to support a hypothesis of common alleles of large effect on foraging behavior in nature. Genome-wide association does support a role for natural variation at several other loci, including the sulfateless gene, though these associations should be considered preliminary until validated with a larger sample size.

Evolutionary Biology↗

bioRxiv2014

Different profile of transcriptome between wheat Yunong 201 and its high-yield mutant Yunong 3114

Wheat is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of wheat grain (as well as rice, maize and other grains) will be critical. Modern technologies are utilized to assist breeding programs. Such as the transcriptome sequencing, which greatly improves our genetic understanding, provides a platform for functional genomics research on crops. Herein, to get an overview of transcriptome characteristics of Yunong 3114, which is screened from the EMS mutagenized population of, a high quality Chinese winter noodle wheat, due to its different plant architecture as well as larger kernel size and higher grain weight, a high-throughput RNA sequencing based on next generation sequencing technology (Illumina) were performed. These unigenes were annotated by Blastx alignment against the NCBI non-redundant (nr), Clusters of orthologous groups (COG), gene orthology (GO), and the Kyoto Encyclopedia of Genesand Genomes (KEGG) databases. The 90.96% of the unigenes matched with protein in the NCBI nr database. Functional analysis identified that changes in several GO categories, including recognition of pollen, apoptotic process, defense response, receptor activity, protein kinase activity, DNA integration and so forth, played crucial roles in the high-yield characteristics of the mutant. Real-time PCR analysis revealed that the recognition of pollen related gene GsSRK is significantly up-regulated in Yunong 3114. In addition, alternative splicing (AS) analysis results indicated that mutation influence AS ratio, especially the retained introns, including the pollen related genes. Furthermore, the digital gene expression spectrum (DGE) profiling data provides comprehensive information at the transcriptional level that facilitates our understanding of the molecular mechanisms of various physiological aspects including development and high-yield of wheat. Together, these studies substantially increase our knowledge of potential genes and pathways for the genetic improvement of wheat and provide new insights into the yield and breeding strategies.

Plant Biology↗

bioRxiv2014

Differential stoichiometry among core ribosomal proteins

Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass-spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

Systems Biology↗

bioRxiv2014

Artificially inducing close apposition of endoplasmic reticulum and mitochondria induces mitochondrial fragmentation.

Cycles of mitochondrial fission and fission are essential for normal cell physiology. Defects in the machinery controlling these processes lead to neurodegenerative disease. While we are beginning to understand the machinery that drives fission, our knowledge of the spatial and temporal control of this event is lacking. Here we use a rapamycin-inducible heterodimerization system comprising both ER and mitochondrial transmembrane components to bring the ER membrane into close physical proximity with mitochondria. We show that this artificial apposition of membranes is sufficient to cause rapid mitochondrial fragmentation. Resulting mitochondrial fragments are shown to be distinct entities using fluorescence recovery after photobleaching. We also show that these fragments retain a mitochondrial membrane potential. In contrast, inducible tethering of the peripheral ER exit site protein TFG does not cause mitochondrial fragmentation suggesting that very close apposition of the two membranes is required.

Cell Biology↗

bioRxiv2014

Testing the Toxicofera: comparative reptile transcriptomics casts doubt on the single, early evolution of the reptile venom system

Background The identification of apparently conserved gene complements in the venom and salivary glands of a diverse set of reptiles led to the development of the Toxicofera hypothesis the idea that there was a single, early evolution of the venom system in reptiles. However, this hypothesis is based largely on relatively small scale EST-based studies of only venom or salivary glands and toxic effects have been assigned to only some of these putative Toxcoferan toxins in some species. We set out to investigate the distribution of these putative venom toxin transcripts in order to investigate to what extent conservation of gene complements may reflect a bias in previous sampling efforts. Results We have carried out the first large-scale test of the Toxicofera hypothesis and found it lacking in a number of regards. Our quantitative transcriptomic analyses of venom and salivary glands and other body tissues in five species of reptile, together with the use of available RNA-Seq datasets for additional species shows that the majority of genes used to support the establishment and expansion of the Toxicofera are in fact expressed in multiple body tissues and most likely represent general maintenance or housekeeping genes. The apparent conservation of gene complements across the Toxicofera therefore reflects an artefact of incomplete tissue sampling. In other cases, the identification of a non-toxic paralog of a gene encoding a true venom toxin has led to confusion about the phylogenetic distribution of that venom component. Conclusions Venom has evolved multiple times in reptiles. In addition, the misunderstanding regarding what constitutes a toxic venom component, together with the misidentification of genes and the classification of identical or near-identical sequences as distinct genes has led to an overestimation of the complexity of reptile venoms in general, and snake venom in particular, with implications for our understanding of (and development of treatments to counter) the molecules responsible for the physiological consequences of snakebite.

Evolutionary Biology↗

bioRxiv2014

Burrow architectural types of the Atlantic ghost crab, Ocypode quadrata (Fabricius, 1787) (Brachyura: Ocypodidae), in Brazil

A broad range of aspects from paleontology to physiology of the ghost crabs Ocypode quadrata have been studied worldwide. These crabs have been used as ecological indicators of the levels of anthropogenic impacts on sandy beaches. Our aim is to report the variety of burrow architecture types constructed by ghost crabs Ocypode quadrata on beaches of Maceio, Brazil. We found 20 types of burrows that differ in shape (number of axes, number of openings, orientation of blind end, number of branches). The slash-shaped burrows (type C) were the most frequent shape, followed by types K (spiral) and E (Y-shaped). Type C also showed the largest opening diameter and length ranges. Burrow types F, J, P, S and T were the least frequent. The G-test for goodness of fit to a time-independent uniform frequency distribution (G = 417.61; d.f. = 18; p < 0.005) reject the hypothesis that burrow types are constructed randomly (uniform distribution). The dominance of type C burrows and other simple-type burrows over more elaborate types indicates preference for simplicity.

Ecology↗

bioRxiv2014

Using Polysome Isolation with Mechanism Alteration to Uncover Transcriptional and Translational Dynamics in Key Genes

What does it mean when we say a cells biochemistry is regulated during changes to the phenotype? While there are a plethora of potential mechanisms and contributions to the final outcome, a more tractable approach is to examine the dynamics of mRNA. This way, we can assess the contributions of both known and unknown decay and aggregation processes for maintaining levels of gene product on a gene-by-gene basis. In this extended protocol, drug treatments that target specific cellular functions (termed mechanism disruption) can be used in tandem with mRNA extraction from the polysome to look at the dynamics of mRNA levels associated with transcription and translation at multiple stages during a physiological perturbation. This is accomplished through validating the polysome isolation method in human cells and comparing fractions of mRNA for each experimental treatment at multiple points in time. First, three different drug treatments corresponding to the arrest of various cellular processes are administered to populations of human cells. For each treatment, the transcriptome and translatome are compared directly at different time points by assaying both cell-type specific and non-specific genes. There are two findings of note. First, extraction of mRNA from the polysome and comparison with the transcriptome can yield interesting information about the regulation of cellular mRNA during a functional challenge to the cell. In addition, the conventional application of such drugs to assess mRNA decay is an incomplete picture of how severely challenged or senescent cells regulate mRNA in response. This extended protocol demonstrates how the gene- and process-variable degradation of mRNA might ultimately require investigations into the course-grained dynamics of cellular mRNA, from transcription to ribosome.

Cell Biology↗

bioRxiv2014

THE GENETIC LANDSCAPE OF TRANSCRIPTIONAL NETWORKS IN A COMBINED HAPLOID/DIPLOID PLANT SYSTEM

Heritable variation in gene expression is a source of evolutionary change and our understanding of the genetic basis of expression variation remains incomplete. Here, we dissected the genetic basis of transcriptional variation in a wild, outbreeding gymnosperm (Picea glauca) according to linked and unlinked genetic variants, their allele-specific (cis) and allele non-specific (trans) effects, and their phenotypic additivity. We used a novel plant system that is based on the analysis of segregating alleles of a single self-fertilized plant in haploid and diploid seed tissues. We measured transcript abundance and identified transcribed SNPs in 66 seeds with RNA-seq. Linked and unlinked genetic effects that influenced expression levels were abundant in the haploid megagametophyte tissue, influencing 48% and 38% of analyzed genes, respectively. Analysis of these effects in diploid embryos revealed that while distant effects were acting in trans consistent with their hypothesized diffusible nature, local effects were associated with a complex mix of cis, trans and compensatory effects. Most cis effects were additive irrespective of their effect sizes, consistent with a hypothesis that they represent rate-limiting factors in transcript accumulation. We show that trans effects fulfilled a key prediction of Wright s physiological theory, in which variants with small effects tend to be additive and those with large effects tend to be dominant/recessive. Our haploid/diploid approach allows a comprehensive genetic dissection of expression variation and can be applied to a large number of wild plant species.

Genomics↗

bioRxiv2014

Side-binding proteins modulate actin filament dynamics

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of the filament to polymerize and depolymerize at its ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. Here, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by proteins that bind to the lateral filament surface. We also show that the less dynamic end, called the pointed-end, has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of filament flexibility and Brownian dynamics simulations suggest that the observed kinetic diversity arises from structural alteration. Tuning filament kinetics by exploiting the natural malleability of the actin filament structure may be a ubiquitous mechanism to generate the rich variety of observed cellular actin dynamics.

Biophysics↗

bioRxiv2014

Genome sequencing of the perciform fish Larimichthys crocea provides insights into stress adaptation

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results provide novel insights into the mechanisms of fish adaptation and response to hypoxia and air exposure.

Genomics↗

bioRxiv2014

Coordinated Evolution of Influenza A Surface Proteins

Surface proteins hemagglutinin (HA) and neuraminidase (NA) of the human influenza A virus evolve under selection pressure to escape the human adaptive immune response and antiviral drug treatments. In addition to these external selection pressures, some mutations in HA are known to affect the adaptive landscape of NA, and vice versa, because these two proteins are physiologically interlinked. However, the extent to which evolution of one protein affects the evolution of the other is unknown. Here we develop a novel phylogenetic method for detecting the signatures of such genetic interactions between mutations in different genes, that is, inter-gene epistasis. Using this method, we show that influenza surface proteins evolve in a coordinated way, with substitutions in HA affecting substitutions in NA and vice versa, at many sites. Of particular interest is our finding that the oseltamivir-resistance mutations in NA in subtype H1N1 were likely facilitated by prior mutations in HA. Our results illustrate that the adaptive landscape of a viral protein is remarkably sensitive to its genomic context and, more generally, imply that the evolution of any single protein must be understood within the context of the entire evolving genome.

Evolutionary Biology↗

bioRxiv2014

Quantifying and understanding reproductive allocation schedules in plants: a lifetime of decisions

O_LIA plant's reproductive allocation (RA) schedule describes the fraction of surplus energy allocated to reproduction as it increases in size. RA schedules distinguish between energy allocated to different tissue types and thus links to a key physiological trade-off in an organism's functioning and life history. While theorists have adopted RA schedules as an elegant connection between life history and energy allocation, little is known about RA schedules in real vegetation.\nC_LIO_LIHere we review what is known about RA schedules for woody plants using studies either directly quantifying RA, or which collected data from which the shape of an RA schedule can be inferred.\nC_LIO_LIWe find that RA schedules vary considerably across species: some species abruptly shift all resources from growth to reproduction (the \"big-bang\" strategy); most others gradually shift resources into reproduction, but under a variety of graded schedules (\"partial bang\", \"asymptotic\", \"gradual\", and \"declining\"). Available data suggest the maximum fraction of energy allocated to production ranges from 0.1 to 1 and that shorter-lived species have higher initial RA and increase their RA more quickly than do longer-lived species.\nC_LIO_LI Synthesis Available data suggests a wide variety of RA schedules exist among plant species. The collection of more data on RA, and not only on easy-to-measure proxies such as maximum height, would enable a tighter integration between theory and observation in plant ecology.\nC_LI

Ecology↗

bioRxiv2014

Amino acid and carbohydrate tradeoffs by honey bee nectar foragers and their implications for plant-pollinator interactions

Honey bees are important pollinators, requiring floral pollen and nectar for nutrition. Nectar is rich in sugars, but contains additional nutrients, including amino acids (AAs). We tested the preferences of free-flying foragers between 20 AAs at 0.1% w/w in sucrose solutions in an artificial meadow. We found consistent preferences amongst AAs, with essential AAs preferred over nonessential AAs. The preference of foragers correlated negatively with AA induced deviations in pH values, as compared to the control. Next, we quantified tradeoffs between attractive and deterrent AAs at the expense of carbohydrates in nectar. Bees were attracted by phenylalanine, willing to give up 84 units sucrose for 1 unit AA. They were deterred by glycine, and adding 100 or more units of sucrose could resolve to offset 1 unit AA. In addition, we tested physiological effects of AA nutrition on forager homing performance. In a no-choice context, caged bees showed indifference to 0.1% proline, leucine, glycine or phenylanaline in sucrose solutions. Furthermore, flight tests gave no indication that AA nutrition affected flight capacity directly. In contrast, low carbohydrate nutrition reduced the performance of bees, with important methodological implications for homing studies that evaluate the effect of substances that may affect imbibition of sugar solution. In conclusion, low AA concentrations in nectar relative to pollen suggest a limited role in bee nutrition. Most of the 20 AAs evoked a neutral to a mild deterrent response in bees, thus it seems unlikely that bees respond to AAs in nectar as a cue to assess nutritional quality. Nonetheless, free choice behavior of foraging bees is influenced, for instance by phenylalanine and glycine. Thus, AAs in nectar may affect plant-pollinator interactions and thereby exhibit a selective pressure on the flora in the honey bee habitat.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC=\"FIGDIR/small/008516_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@8fde04org.highwire.dtl.DTLVardef@138fdd3org.highwire.dtl.DTLVardef@a05775org.highwire.dtl.DTLVardef@cde793_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS- Amino acids in artificial nectar elicit preferences from honey bee foragers\n- Amino acid identity, pH, and essentiality explain preferences of bees\n- A honey bee forager is willing to pay a premium of carbohydrates for amino acids\n- Carbohydrate nutritional state affects flight performance of foraging bees

Animal Behavior and Cognition↗

bioRxiv2014

Myoinhibitory peptide regulates feeding in the marine annelid Platynereis

During larval settlement and metamorphosis, marine invertebrates undergo changes in habitat, morphology, behavior and physiology. This change between life-cycle stages is often associated with a change in diet or a transition between a non-feeding and a feeding form. How larvae regulate changes in feeding during this life cycle transition is not well understood. Neuropeptides are known to regulate several aspects of feeding, such as food search, ingestion and digestion. The marine annelid Platynereis dumerilii has a complex life cycle with a pelagic non-feeding larval stage and a benthic feeding postlarval stage, linked by the process of settlement. The conserved neuropeptide myoinhibitory peptide (MIP) is a key regulator of larval settlement behavior in Platynereis. Whether MIP also regulates the initiation of feeding, another aspect of the pelagic-to-benthic transition in Platynereis, is currently unknown. Here, we explore the contribution of MIP to feeding in settled Platynereis postlarvae. We find that MIP is expressed in the gut of developing larvae in sensory neurons that densely innervate the foregut and hindgut. Activating MIP signaling by synthetic neuropeptide addition causes increased gut peristalsis and more frequent pharynx extensions leading to increased food intake. Conversely, morpholino-mediated knockdown of MIP expression inhibits feeding. In the long-term, treatment of Platynereis postlarvae with synthetic MIP increases growth rate and results in earlier cephalic metamorphosis. Our results show that MIP activates ingestion and digestion in Platynereis postlarvae. MIP is expressed in sensory-neurosecretory cells of the digestive system indicating that following larval settlement, feeding is initiated by a direct sensory mechanism. This is similar to the mechanism by which MIP induces larval settlement. The pleiotropic roles of MIP may thus have evolved by redeploying the same signaling mechanism in different aspects of a life cycle transition.

Zoology↗

bioRxiv2014

Deciphering the genetic and transcriptional basis of cross-stress responses in Escherichia coli under complex evolutionary scenarios

A tantalizing question in microbial physiology is the inter-dependence and evolutionary potential of cellular stress response across multiple environmental dimensions. To address this question, we comprehensively characterized the cross-stress behavior of wild-type and evolved Escherichia coli populations in five abiotic stresses (n-butanol, osmotic, alkaline, acidic, and oxidative) by performing genome-scale genetic, transcriptional and growth profiling, thus identifying 18 cases of cross-stress protection and one case of cross-stress vulnerability. We identified 18 cases of cross-stress protection and one case of cross-stress vulnerability, along with core and stress-specific networks. We tested several hypotheses regarding the effect of the stress order, stress combinations, mutation reversal and varying environments to the evolution and final cellular fitness of the respective populations. Our results argue of a common systems-level core of the stress response with several crucial implicated pathways that include metal ion binding and glycolysis/gluconeogenesis that is further complemented by a stress-specific expression program.

Evolutionary Biology↗

bioRxiv2014

A novel computational approach for genome-wide prediction of small RNAs in bacteria

Small regulatory RNAs (sRNAs) are the most abundant post-transcriptional regulators in bacteria. They serve ubiquitous roles that control nearly every aspects of bacterial physiology. Identification of important features from sRNAs sequences will guide the computational prediction of new sRNA sequences for a better understanding of the pervasive sRNA-mediated regulation in bacteria. In this study, we have performed systematic analyses of many sequence and structural features that are possibly related to sRNA properties and identified a subset of significant features that effectively discriminate sRNAs sequences from random sequences. we then used a neural network model that integrated these subfeatures on unlabeled testing datasets, and it had achieved a 92.2% recall and 89.8% specificity. Finally, we applied this prediction model for genome-wide identification of sRNAs-encoded genes using a sliding-window approach. We recovered multiple known sRNAs and hundreds of predicted new sRNAs. These candidate novel sRNAs deserve extensive study to better understand the sRNA-mediated regulatory network in bacteria.

Bioinformatics↗

bioRxiv2014

A systematic simulation of the effect of salicylic acid on sphingolipid metabolism

AbstractThe phytohormone salicylic acid (SA) affects plant development and defense responses. Recent studies revealed that SA is also involved in the regulation of sphingolipid metabolism, but the details of this regulation remain to be explored. Here, we use in silico Flux Balance Analysis (FBA) with published microarray data to construct a whole-cell simulation model, including 23 pathways, 259 reactions and 172 metabolites, to predict the alterations in flux of major sphingolipid species after treatment with exogenous SA. This model predicts significant changes in fluxes of certain sphingolipid species after SA treatment, changes that likely trigger downstream physiological and phenotypic effects. To validate the simulation, we used isotopic non-stationary metabolic flux analysis to measure sphingolipid contents and turnover rate in Arabidopsis thaliana seedlings treated with SA or the SA analog benzothiadiazole (BTH). The results show that both SA and BTH affect sphingolipid metabolism by not only concentration of certain species, but also the optimal flux distribution and turnover rate of sphingolipid contents. Our strategy allows us to formally estimate sphingolipid fluxes on a short time scale and gives us a systemic view of the effect of SA on sphingolipid homeostasis.

Plant Biology↗

bioRxiv2014

Gene expression associated with white syndromes in a reef building coral, Acropora hyacinthus.

BackgroundCorals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.\n\nResultsTag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.\n\nConclusionsSimilarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.

Genomics↗