A systematic effort to identify the natural ecology of Pristionchus nematodes revealed species-specific host preferences for several beetle species, including the oriental beetle found in Japan and northeastern United States. Although biologists have been aware of such insect-nematode associations for some time, the details of these interactions remain largely unknown. In Pristionchus pacificus, the cGMP dependent protein kinase,
Ppa-egl-4 is an important genetic modifier of insect pheromone reception. Upregulation of
Ppa-egl-4 transcripts depends on intracellular cGMP levels and is highly variable among wild isolates from diverse host ranges. In particular, chemoattraction to the Oriental beetle sex pheromone is very strong in the Washington isolate (PS1843) but is elicited in the reference isolate California (PS312) only after a brief exogenous cGMP treatment. To obtain additional factors involved in insect pheromone attraction, we performed forward genetic screens in P. pacificus for mutants that do not show attraction toward the oriental beetle pheromone (Z-7-tetradecen-2-one, ZTDO) in both the natural isolates. From our behavior-based screens, we isolated two Oriental Beetle pheromone Insensitive mutants,
obi-1 and
obi-3 from the California strain, as well as two Washington obi's,
wobi-2 and
wobi-4. The chemosensory phenotype of
obi-1 and
obi-3 is strongly specific for ZTDO and does not affect the chemoattraction toward other known P. pacificus attractants such as the plant volatile b-caryophyllene and the moth pheromone ETDA. The chemosensory profiles of
wobi-2 and
wobi-4 are also specific for ZTDO but show noticeable decrease in two other odors as well. We focused on the molecular cloning of
obi-1 based on its strong and specific ZTDO attraction defect. In addition to the chemosensory defect, adult
obi-1 hermaphrodites are ~20 percent longer than wildtype and moderately egg-laying defective. Using molecular SSLP markers, we were able to delineate the
obi-1 lesion to a ~428 kb region represented by the ends of Supercontigs 1 and 15 on chromosome I. We further identified all polymorphic sites between PS312 and
obi-1 in both Supercontigs by whole genome sequencing (Illumina) and found a single basepair deletion in the predicted coding region of a gene homologous to the C. elegans C06G1.1, an uncharacterized protein with a lipid-binding motif. Confirmation of this novel gene as a mediator of EGL-4-dependent insect pheromone sensing by transgenic rescue and RNAi knockdown is currently ongoing.