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The characterization and biological control potential of an endemic entomopathogenic nematode and its symbiotic bacterium through behavioural, molecular and genomic approaches

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
Date January 2016
CreatorsSoobramoney, Lee-Anne Odelle
Source SetsSouth African National ETD Portal
Detected LanguageEnglish
FormatOnline resource (xv, 173 leaves), application/pdf

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