Surveys for entomopathogenic bacteria and their associated nematode hosts were conducted locally (University of Bath campus) and across southern England. Sampling involved trialing a novel Android app. (Epicollect) to manage sample collection data. Galleria larvae were used to bait UK soil samples. Insects which became infected were placed on White traps to collect any emerging nematodes, from which bacteria were isolated. Bacteria were also isolated from the haemolymph of any infected larvae. Bacterial isolates were classified on the basis of 16s rDNA and recA gene sequences. Serratia proteamaculans-like strains dominated the samples, and Multilocus sequence analysis (MLSA) was developed for the characterization of these Serratia isolates. We determined the sequences of (350-450-bp) fragments from five housekeeping genes of 84 isolates of Serratia proteamaculans. MLSA was shown to be effective for distinguishing closely related strains found in the insects’ haemolymph and from different nematodes. goeBURST was used to visualize the relationships between the STs, and the data showed a high level of discrimination, resolving 69 STs from the 84 isolates. In addition, the data derived from this study were represented in a phylogenetic network using the Splits Tree-network methods, to show the rate of recombination within and between the genes. From a total of 256 infected Galleria 23.04% were nematode positive. The nematodes were identified based on 18S rDNA 19 isolates were close relatives of the species Pristionchus entomophaga and Diplogasteriodes magnus (Diplogastridae). A further 16 isolates were more closely related to Steinernema glaseri (Steinernematidae). All three nematode types were isolated from diverse habitats and soil types, but were isolated more frequently in cold seasonal conditions. The bacterial sequence data suggest that the nematode- associated strains of bacteria belong to specific clades, distinct from the free living infective strains, which hints at ecological diversity within the S. proteamaculans population. Two of the Serratia proteamaculans-like strains had been chromosomally labeled with GFP to confirm the specifics of their association with the nematode hosts. The associated S. proteamaculans-like isolates isolated from Bath and Chepstow soils were examined further for their pathogenicity to Galleria mellonella and Manduca sexta larvae. Serratia Bath isolates, isolated from Pristionchus were more virulent toward both insect hosts than the Serratia from the Chepstow isolates associated with Steinernema nematodes. This suggests that host specificity may play important role in the virulence of the strain.

Wir fanden konservierte molekulare Signaturen der Regulation durch Δ7-DA und Ascarosid bei Dauer- und infektiösen Larven. Danach wurde die hohe Konservierung durch unsere Analyse in Dauer- und Postdauer-Stadium zwischen den zwei nah verwandten freilebenden Arten C. elegans und C. briggsae identifiziert. Das heißt, dass die relative Veränderung auf mRNA- oder Protein- Ebene zwischen zwei Arten stark korreliert ist. Aber die relative Veränderung innerhalb derselben Art zeigt keine hochgradige Korrelation zwischen mRNA- und Protein-Ebene. Unsere Ergebnisse zeigen in C. elegans Dauerlarven die signifikante Reduzierung der RNA-Mengen in 20 Stoffwechselwegen. Im Gegensatz dazu speicherten Dauerlarven reichlich RNA-Mengen in GO Termen wie Ribosome und Aminoacyl-tRNA biosynthesis. Auf Protein-Ebene sind die Stoffwechselwege von Proteinsynthese und Proteinverarbeitung im endoplasmatischen Retikulum in Dauerlarven herunterreguliert und GO Terme wie Lysosome sind hochreguliert. Durch die Zeitreihenanalyse der Proteom-Remodellierung der molekularen Signaturen beim Austritt aus dem Dauer-Stadium fand wir, dass GO Terme wie metal ion binding signifikant herunterreguliert sind und der Proteinabbau hochreguliert ist. Unsere Ergebnisse vom pSILAC Experiment deuten an, dass die Proteine für Energieerzeugung und Chaperone/Proteinfaltung beim Daueraustritt schnell verbraucht sind und wieder hergestellt werden. Zum Schluss haben wir als Erste den popomR-Assay in C. elegans etabliert und ein Screening der vermeintlichen Proteinbindestellen auf poly-A-RNA durchgeführt, um in der Zukunft die konservierten Mechanismen der post-transkriptionellen Regulation durch RBPs im Dauer-Stadium zu analysieren. / We found the conservation of molecular signatures by regulating with Δ7-DA and Ascarosid in dauer larvae and infective larvae. Then by our comparative analysis, the high degree of conservation between two closely related free-living species C. elegans and C. briggsae was identified in dauer and post-dauer stages. This means that the relative changes are strongly correlated on the mRNA or the protein level between two species. But the relative changes in the same species don’t show any strong correlation between the mRNA and the protein levels. Our results showed a significantly reduced amount of RNA in 20 metabolic pathways in C. elegans dauer larvae. In contrast, dauer larvae stored a large amount of RNA in GO terms such as ribosome and aminoacyl-tRNA biosynthesis. On the protein level, the metabolic pathways of protein synthesis and protein processing in endoplasmic reticulum were downregulated in dauer larvae and the term of lysosome was up-regulated. Due to time course analysis for proteome remodeling of molecular signatures during exit process from dauer stage, we found that GO terms such as metal ion binding were significantly downregulated during dauer exit and at the same time the protein degradation was up-regulated. Our results of pSILAC experiment suggest that the proteins for energy generation and chaperone/protein folding are quickly spent and rebuilded during dauer exit. Finally, we were the first to establish the popomR assay in C. elegans and performed a screening of the putative protein binding sites on poly-A RNA to analyze the conserved mechanisms of post-transcriptional regulation by RBPs in dauer larvae in the future.