Entomopathogenic nematodes (EPNs) (genera Heterorhabditis and Steinernema) kill their invertebrate hosts with the aid of a mutualistic bacterium. The bacteria (Xenorhabdus spp. for steinernematids and Photorhabdus spp. for heterorhabditids) are primarily responsible for killing the host and providing the nematodes with nutrition and defense against secondary invaders. Photorhabdus is a Gram-negative bacterium in the Enterobacteriaceae family with high virulence towards their insect hosts. To achieve high mortality rates Photorhabdus produces a variety of virulence factors such as toxins, lipases, proteases, secretion systems, and fimbriae. EPNs are amenable to laboratory rearing and mass production for biocontrol applications against insects using in vivo or in vitro methods; however, in vitro liquid culture is considered to be the most efficient. In this method the symbiotic bacteria are cultured prior to the addition of their partner EPN. This can leave the bacteria susceptible to a number of problems such as genetic drift and inadvertent selection. Regardless of the culture method the symbiotic bacteria exhibit trait deterioration or changes due to laboratory rearing. This project had three primary aims: 1) investigate the role of nutrition in trait deterioration, 2) examine virulence evolution using a phylogenetic context, and 3) identify genes that are necessary for survival and virulence inside the insect host. Prior to studying these objectives we first determined the optimal conditions for growing and counting viable cells of Photorhabdus. We discovered that growth is enhanced by the addition of pyruvate to growth media. To determine the role of nutrition in trait deterioration we repeatedly sub-cultured Photorhabdus in three different media types. Throughout this study we found that, in contrast to previous studies, trait deterioration does not always happen and the environment influences trait deterioration. Furthermore, based on our phylogenetic studies we found that Photorhabdus spp. are evolving to an increase in insect virulence. Lastly, using Tn-seq we determined a list of 84 genes that are needed for efficient virulence inside the insect host and provide suggestions for ongoing research efforts.



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Life Sciences; Biology



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entomopathogenic nematodes, Photorhabdus, trait deterioration, nutrition, Tn-seq, evolution

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Biology Commons