15 research outputs found
Hierarchical Alignment and Full Resolution Pattern Recognition of 2D NMR Spectra: Application to Nematode Chemical Ecology
Ascaroside Expression in Caenorhabditis elegans Is Strongly Dependent on Diet and Developmental Stage
Background:
The ascarosides form a family of small molecules that have been isolated from cultures of the nematode Caenorhabditis elegans. They are often referred to as “dauer pheromones” because most of them induce formation of long-lived and highly stress resistant dauer larvae. More recent studies have shown that ascarosides serve additional functions as social signals and mating pheromones. Thus, ascarosides have multiple functions. Until now, it has been generally assumed that ascarosides are constitutively expressed during nematode development.
Methodology/Principal Findings:
Cultures of C. elegans were developmentally synchronized on controlled diets. Ascarosides released into the media, as well as stored internally, were quantified by LC/MS. We found that ascaroside biosynthesis and release were strongly dependent on developmental stage and diet. The male attracting pheromone was verified to be a blend of at least four ascarosides, and peak production of the two most potent mating pheromone components, ascr#3 and asc#8 immediately preceded or coincided with the temporal window for mating. The concentration of ascr#2 increased under starvation conditions and peaked during dauer formation, strongly supporting ascr#2 as the main population density signal (dauer pheromone). After dauer formation, ascaroside production largely ceased and dauer larvae did not release any ascarosides. These findings show that both total ascaroside production and the relative proportions of individual ascarosides strongly correlate with these compounds' stage-specific biological functions.
Conclusions/Significance:
Ascaroside expression changes with development and environmental conditions. This is consistent with multiple functions of these signaling molecules. Knowledge of such differential regulation will make it possible to associate ascaroside production to gene expression profiles (transcript, protein or enzyme activity) and help to determine genetic pathways that control ascaroside biosynthesis. In conjunction with findings from previous studies, our results show that the pheromone system of C. elegans mimics that of insects in many ways, suggesting that pheromone signaling in C. elegans may exhibit functional homology also at the sensory level. In addition, our results provide a strong foundation for future behavioral modeling studies
<sup>13</sup>C NMR Metabolomics: INADEQUATE Network Analysis
The many advantages of <sup>13</sup>C NMR are often overshadowed
by its intrinsically low sensitivity. Given that carbon makes up the
backbone of most biologically relevant molecules, <sup>13</sup>C NMR
offers a straightforward measurement of these compounds. Two-dimensional <sup>13</sup>C–<sup>13</sup>C correlation experiments like INADEQUATE
(incredible natural abundance double quantum transfer experiment)
are ideal for the structural elucidation of natural products and have
great but untapped potential for metabolomics analysis. We demonstrate
a new and semiautomated approach called INETA (INADEQUATE network
analysis) for the untargeted analysis of INADEQUATE data sets using
an <i>in silico</i> INADEQUATE database. We demonstrate
this approach using isotopically labeled <i>Caenorhabditis elegans</i> mixtures
Metabolomics and Natural-Products Strategies to Study Chemical Ecology in Nematodes
Synopsis This review provides an overview of two complementary approaches to identify biologically active compounds for studies in chemical ecology. The first is activity-guided fractionation and the second is metabolomics, particularly focusing on a new liquid chromatography-mass spectrometry-based method called isotopic ratio outlier analysis. To illustrate examples using these approaches, we review recent experiments using Caenorhabditis elegans and related freeliving nematodes
Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans
We demonstrate the global metabolic
analysis of Caenorhabditis elegans stress
responses using a mass-spectrometry-based
technique called isotopic ratio outlier analysis (IROA). In an IROA
protocol, control and experimental samples are isotopically labeled
with 95 and 5% <sup>13</sup>C, and the two sample populations are
mixed together for uniform extraction, sample preparation, and LC-MS
analysis. This labeling strategy provides several advantages over
conventional approaches: (1) compounds arising from biosynthesis are
easily distinguished from artifacts, (2) errors from sample extraction
and preparation are minimized because the control and experiment are
combined into a single sample, (3) measurement of both the molecular
weight and the exact number of carbon atoms in each molecule provides
extremely accurate molecular formulas, and (4) relative concentrations
of all metabolites are easily determined. A heat-shock perturbation
was conducted on C. elegans to demonstrate
this approach. We identified many compounds that significantly changed
upon heat shock, including several from the purine metabolism pathway.
The metabolomic response information by IROA may be interpreted in
the context of a wealth of genetic and proteomic information available
for C. elegans. Furthermore, the IROA
protocol can be applied to any organism that can be isotopically labeled,
making it a powerful new tool in a global metabolomics pipeline
Activity guided fractionation of the <i>S. feltiae</i> dispersal pheromone.
<p>(A) Reverse phase (C18) chromatography of insect cadaver extract. The image represents two independent experiments. The estimated physiologically relevant concentration was used in the assays; Frc, fraction. (B) Testing physiologically relevant concentration of ascarosides found in Frc A (ascr#9, 40.3 pmol/µl and ascr#11, 1.3 pmol/µl). Image represents three experiments. (C) Ascr#2 and ascr#9 are structural analogs. Natural and synthetic ascr#9 were tested in combination with fraction B and C. Image represents four experiments. (D) Structure of ascr#11. It (1.3 pmol/µl) is also sufficient to cause dispersal in combination with fractions B and C. Image represents three experiments.</p