38th Annual Meeting of the Society for Invertebrate Pathology

The nematode Heterorhabditis bacteriophora is both an insect parasite and a host in an obligate mutualism with the insect pathogenic bacterium, Photorhabdus luminescens. The tripartite interactions between nematode, bacterium and insect involves several interesting attributes e.g.: 1) symbiont specific colonization of the intestinal lumen of the infective juvenile (IJ) stage nematode, 2) hunting, infection and regurgitation of P. luminescens bacteria by IJs in hemolymph, and 3) symbiont dependent growth and reproduction of the nematode. Although H. bacteriophora and Caenorhabditis elegans are both in the family Rhabditidae, genetic techniques were underdeveloped and not applied for the study of symbiosis. Here we show the successful application of RNAi in H. bacteriophora. First larval stage nematodes were soaked with in vitro transcribed dsRNA corresponding to H. bacteriophora orthologs of cct-2 (HSP60 chaperonin), daf-21 (HSP90), icd-1 (anti-apoptotic BTF3 transcription factor), 2 ribosomal biogenesis genes (W01G7.3, W01B11.3) and a guanine nucleotide binding protein (K04D7.1) which had strong phenocopies in C. elegans. For H. bacteriophora, the penetrance of RNAi was high (70-100%) except for K04D7.1 that had moderate penetrance (~50%). The resulting phenocopies were similar but not identical in H. bacteriophora and C. elegans. Forward genetics using the mutagen EMS was moderately successful in H. bacteriophora resulting in 2 alleles corresponding to “unc-22” or “twitchin” and several egl (egg laying defective) and dpy (dumpy) phenotypes. We are applying forward and reverse genetics to elucidate host genes involved in symbiont specific colonization of the nematode gut and growth and development as well as insect parasitism.