A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology in
fulfilment for requirements for Doctor of Philosophy Degree.
February 2016 / The use of synthetic chemical pesticides has several negative implications for the Agricultural
industry, which include the development of resistance to the insecticides, crop contamination and
the killing of non-target insects. This has brought many scientists in the field of nematology and
entomology to investigate biological control agents which can help solve identified challenges and
these biocontrol agents have also included entomopathogenic nematodes. The majority of
entomopathogenic nematodes species that have been isolated belong to Heterorhabditids and
Steinernematids which act as vectors for insect pathogenic bacteria species belonging to the genera,
Photorhabdus and Xenorhabdus, respectively. However, other species of nematodes, one of which
includes a strain of Caenorhabditis briggsae, have also been shown to act as a vector for an insect
pathogenic strain of Serratia marcescens. Oscheius sp. TEL-2014 EPNs have been observed to act
as vectors for insect pathogenic bacteria belonging to the genus Serratia. In this study a novel
insect pathogenic Serratia sp. strain TEL was isolated from the gut of infective juveniles belonging
to a species of Oscheius sp. TEL-2014. Next generation sequencing of the bacteria was conducted
by generating genomic DNA paired-end libraries with the Nextera DNA sample preparation kit
(Illumina) and indexed using the Nextera index kit (Illumina). Paired-end (2 × 300 bp) sequencing
was performed on a MiSeq Illumina using the MiSeq reagent kit v3 at the Agricultural Research
Council Biotechnology Platform. Quality control and adapter trimming was performed and the
genome was assembled using SPADES. 19 contigs were generated with an average length of
301767 bp and N50 of 200,110 bp. The genome of the Serratia sp. TEL was found to be 5,000,541
bp in size, with a G+C content of 59.1%, which was similar to that of other Serratia species
previously identified. Furthermore, the contigs were annotated using NCBI Prokaryotic Genome
Automatic Annotation Pipeline. Features of the annotated genome included protein encoding
sequence or genes, rRNA encoding genes, tRNA encoding genes, ncRNA sequences and repeat
regions. 4,647 genes were found and 4,495 were protein-coding sequences (CDS). The genome
contains 36 pseudo genes, 2 CRISPR arrays, 13 rRNA genes with five operons (5S, 16S, 23S), 88
tRNAs genes, 15 ncRNA sequences and 9 frameshifted genes. Several genes involved in virulence,
disease, defense, stress response, cell division, motility and chemotaxis were identified. This
genome sequence will allow for the investigation of identified genes and that will be critical in
furthering the understanding of the insect pathogenicity of Serratia sp. strain TEL. Furthermore, it
will provide additional genomic insights about the insect-nematode interactions and thus help us
improve their ability to be used as biological control agents in agricultural industries. Oscheius sp.
TEL-2014 was tested for its entomopathogenicity and it was found that this species was able to
infect and kill two model insects Galleria mellonella and Tenebrio molitor. This new nematode
species brought 100% mortality within 72 h post-exposure in G. mellonella and whereas, within 96
hours in T. molitor. Following morphometrics analysis of Oscheius sp. TEL-2014 it was concluded
that this nematode is described as a novel entomopathogenic nematode species based on its
morphometrics and 18S rRNA gene sequence originality. Whole genome sequencing of Oscheius
sp. TEL-2014 inbred lines (7 and 13) was performed using Illumina Hiseq sequencing system and
paired ends library preparation protocol. Sequencing reads assembled on Velvet resulted in
generation of 75965 contigs (line 7) and 53190 contigs (line 13). Gene prediction tools showed that
proteins involved in gene expression and DNA replication are present in Oscheius sp. TEL-2014.
The draft genome of Oscheius nematodes will support the improvement and initiation of further
studies intended to help us understand the molecular and metabolic processes in this genus.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/20355 |
| Date | 10 May 2016 |
| Creators | Lephoto, Tiisetso Elizabeth |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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