Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca2+ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound

Molecular Time-Course and the Metabolic Basis of Entry into Dauer in Caenorhabditis elegans

When Caenorhabditis elegans senses dauer pheromone (daumone), signaling inadequate growth conditions, it enters the dauer state, which is capable of long-term survival. However, the molecular pathway of dauer entry in C. elegans has remained elusive. To systematically monitor changes in gene expression in dauer paths, we used a DNA microarray containing 22,625 gene probes corresponding to 22,150 unique genes from C. elegans. We employed two different paths: direct exposure to daumone (Path 1) and normal growth media plus liquid culture (Path 2). Our data reveal that entry into dauer is accomplished through the multi-step process, which appears to be compartmentalized in time and according to metabolic flux. That is, a time-course of dauer entry in Path 1 shows that dauer larvae formation begins at post-embryonic stage S4 (48 h) and is complete at S6 (72 h). Our results also suggest the presence of a unique adaptive metabolic control mechanism that requires both stage-specific expression of specific genes and tight regulation of different modes of fuel metabolite utilization to sustain the energy balance in the context of prolonged survival under adverse growth conditions. It is apparent that worms entering dauer stage may rely heavily on carbohydrate-based energy reserves, whereas dauer larvae utilize fat or glyoxylate cycle-based energy sources. We created a comprehensive web-based dauer metabolic database for C. elegans (www.DauerDB.org) that makes it possible to search any gene and compare its relative expression at a specific stage, or evaluate overall patterns of gene expression in both paths. This database can be accessed by the research community and could be widely applicable to other related nematodes as a molecular atlas

Development of a Method to Quantitate Nematode Pheromone for Study of Small-Molecule Metabolism in <i>Caenorhabditis elegans</i>

Pheromones produced by <i>Caenorhabditis elegans</i> are considered key regulators of development, mating, and social behaviors in this organism. Here, we present a rapid mass spectrometry-based method (PheroQu) for absolute <i>qu</i>antitation of nematode <i>phero</i>mones (e.g., daumone 1, 2, and 3) both in <i>C. elegans</i> worm bodies (as few as 20 worms) and in liquid culture medium. Pheromones were separated by ultra performance liquid chromatography and monitored by a positive electrospray ionization detector in the multiple-reaction monitoring mode. The <i>daf-22</i> mutant worms were used as surrogate matrix for calibration, and stable deuterated isotope-containing pheromone was used as internal standard for measuring changes in pheromones in N2 wild-type and other strains under different growth conditions. The worm-body pheromones were extracted by acidified acetonitrile solvent, and the secreted pheromones were extracted from culture medium with solid-phase extraction cartridges. The run time was achieved in less than 2 min. The method was validated for specificity, linearity, accuracy, precision, recovery, and stability. The assay was linear over an amount range of 2–250 fmol, and the limit of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both worm bodies and culture medium. With the PheroQu method, we were able to identify the location of pheromone biosynthesis and determine the changes in different pheromone types synthesized, according to developmental stages and aging process. This method, which is simple, rapid, sensitive, and specific, will be useful for the study of small-molecule metabolism during developmental stages of <i>C. elegans</i>