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A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2016. / The entomopathogenic nematodes (EPNs) have emerged as an important group of insect pests. The EPNs which comprise the Steinernema genus share symbiotic associations with Xenorhabdus bacteria. This research project focused on isolating and characterizing a novel and indigenous EPN isolate with its associated bacteria. The biological control potential of the nematode was investigated in the areas of host infectivity, infective juvenile recovery and progeny yield. These processes were investigated at three different factors. These included time, population size and temperature. The infectious abilities of the symbiotic bacteria were also evaluated without the contributions of the nematode partner at different bacterial doses, time intervals and temperature regimens. The genome of the bacteria was thereafter acquired through whole-genome sequencing and annotation techniques to elucidate the virulence mechanisms and genes involved in temperature adaptation. The species isolated in this investigation was novel. The species shared an 85 % maximum identity to and taxonomically grouped with the species Steinernema khoisanae. The two species shared a common ancestor but the extended branch length of the species under investigation substantiated its novelty. The EPNs infected hosts at different time intervals, population densities and temperature regimens. However, the EPNs performed these processes to different extents. Host mortality significantly increased with time. The EPNs also infected insect hosts at the two experimental temperatures. However, host mortality was higher at the temperature regimen of 20° C and lower at 30° C. Host infections were not significantly different at two tested population densities of 500 and 1000 infective juveniles. The levels of interaction between temperature and time and temperature population density were not statistically significant. The subsequent biological process of recovery was evaluated. The EPNs recovered at both population densities and temperature regimens. The infective juvenile recoveries were statistically insignificant at both population densities and temperature regimens. Since recovery was based on the mere presence of progeny infective juveniles, the percentages were high which contributed to the statistical insignificant findings. This also contributed to the non-significant interaction between population density and temperature. The last biological process investigated was the progeny yield of infective juveniles. The yields were significantly different between both population densities of infective juveniles and temperature regimens. Higher yields were obtained at the temperature regimen of 20° C and 25° C. Lower yields were obtained at 30° C. The unexpected finding was higher progeny yields obtained from the lower population densities of infective juveniles. This contributed to the significant interaction present between population density and temperature. The bacteria were thereafter molecularly characterized. The symbiotic bacteria shared a 99 % sequence similarity to the species Xenorhabdus sp. strain GDc328. It was interesting to observe the infectious abilities of the bacteria without contributions from the EPNs. This study was measured at different bacterial doses, time intervals and temperature regimens. Host mortality was achieved without contributions from the EPN. Host mortality significantly increased with bacterial dose and time. Host mortality was also significantly different between each temperature regimen. Higher mortalities were observed at 30° C and lower mortalities were observed at 20° C. The differences in the performance between the EPN-bacterial partnership and the bacteria alone were attributed to the manner in which adaptation occurred. Since the EPN-bacteria existed as a bi-partite entity, the partners evolved as a bi-partite complex. The bacteria were removed from the symbiosis and cultured individually. External factors may have re-shaped the performance of the bacteria at the different temperature regimens. To further understand the genetic mechanisms of temperature adaptation, host infectivity and symbiosis, the draft whole genome sequence of the bacteria was then acquired. The genome of the bacteria comprised several genes which encoded the flagella system of the bacteria. Also pairs of co-localized toxin-antitoxin genes were discovered. Temperature acclimatization was performed through different cold and heat shock proteins and lastly several molecular chaperones. The studies showed that the species Steinernema spp. and its associated symbiotic bacteria Xenorhabdus sp. strain GDc328 were good bio-pesticide candidates for application against endemic insect pests. / LG2017

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