Thesis

The characterization of obi-1 and obi-3 beetle pheromone insensitive mutants in Pristionchus pacificus nematodes

Nematodes rely on chemosensation for their survival. Through chemosensation nematodes can avoid harmful chemicals, enter into the dauer alternative developmental stage, find food sources, potential mates, and even insect hosts. Pristionchus pacificus (P. pacificus), isolated from Pasadena, California in 1996 have been found to associate with different beetle hosts around the world. In particular, P. pacificus has shown preference in associating with the oriental beetle found in Japan and northeastern United States and is specifically attracted to the beetle's sex pheromone (Z)-7-tetradecen-2-one (ZTDO) in the laboratory. To determine the genes responsible for insect pheromone sensing, two chemosensory mutants were identified in a genetic screen for lack of attraction towards ZTDO after cGMP treatment, named Ppa-obi-1 and Ppa-obi-3 (oriental beetle pheromone insensitive). Protein expression of a recombinant GST-Ppa-OBI-1 (in E. coli) and recombinant GFP-Ppa-OBI-1 (in C. elegans) have been analyzed using western blots. The second aim of my study is to characterize another mutant allele, Ppa-obi-3 (tu405), which has a short body length and coiling phenotype. In C. elegans, reversal rates are known to affect approach behavior in chemotaxis. I determined Ppa-obi-3's reversal rates by counting the frequency of omega turns on and off food and found that Ppa-obi-3 animals reverses significantly more often than wildtype. Interestingly, C. elegans egl-30 mutants show a similar defective locomotion behavior with a higher reversal rate compared to wildtype. Defects in EGL-30 (Gqα) affects the release of acetylcholine from motor neurons needed for normal muscle movement to occur. I found that the associated behavior is further accentuated in liquid medium. Wildtype PS312 shows a thrashing motion in which the head is restricted to a small angle, whereas Ppa-obi-3 coils into a superimposed circle, and head as a result changes directions continuously. This coiling-swimming phenotype was utilized for positional mapping of Ppa-obi-3 on the genetic linkage map. Homozygotes from Ppa-obi-3 x Japan (RS5195) recombinant inbred lines were rough mapped to a 38cM interval of Chromosome I (out of 6 linkage groups) using simple sequence length polymorphism markers. The long-term goal is to determine if Ppa-obi-3 and Ppa-obi-1 constitute components of an unknown signaling pathway, part of the conserved cGMP-dependent G protein signaling pathway, or TRPV channels lipid signaling pathway (PUFA) involved in sensing insect hosts.

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