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

The life-history and infection parameters of the entomopathogenic nematodes Steinernema feltiae (Filipjev)(Nematoda:Rhabditida) and Heterorhahditis megidis (Poinar, Jackson & Klein)(Nematoda:Rhabditida) were examined to provide specific details for the construction of mathematical SI models for biological control of soil insect pests. Laboratory experiments using the Greater Waxmoth, Galleria mellonella as the model host were undertaken to specifically examine the transmission behaviour of infective juvenile nematodes. The proportion of infective juveniles of S. feltiae which infected hosts was dependent on time. Previous studies declared that the proportion of infective juveniles which can infect is static, however, over a period of 5 days most of the infective juveniles infected hosts, demonstrating that the proportion infecting is dynamic. Infection of hosts by both species of nematode was compared using two mathematical representations of the transmission rate. Whereas the most parsimonious form of transmission for H. megidis was the linear Mass Action function, it was evident that, when measured at the individual nematode scale, S. feltiae transmission was non-linear. I postulated that this functional difference is due to the biology of the two species of nematodes. The subsequent effect of including the non-linear response on model predictions were investigated and it was demonstrated that the dynamics of the host nematode interaction became less stable. Spatial models of S. feltiae infection were parameterised from laboratory experiments, and control prediction of these models examined. The horizontal rate of dispersal through sand columns was determined in the presence and absence of hosts. Infective juveniles were found to disperse preferentially towards hosts. The predicted dynamics of pest control using the spatial moqel were highly dependent on the degree of nematode dispersal, host dispersal and the attraction of nematode infective juveniles towards hosts. The overall findings of this thesis have been placed in the context of epidemiological models created elsewhere, and predict that entomopathogenic nematodes may be targeted to specific pest systems with a high degree of success. An understanding of the infection biology of these nematode species is crucial in determining how and when pests may be controlled, and equally importantly, which systems successful control is not predicted.