A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Year: 2016. / Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema have emerged excellent as non-chemical alternatives for control of insect pest population. They have a specific mutualistic symbioses with bacterial symbionts in the genera Photorhabdus and Xenorhabdus, respectively. Native EPN species that are able to tolerate environmental stress including desiccation are of great interest for application. The aim of this study was to isolate indigenous EPN species from soil samples collected from Brits, North West province in South Africa, and to investigate their ability to tolerate desiccation stress. The second aim was to isolate the bacterial symbiont and sequence, assemble and annotate its whole genomic DNA. Insect baiting technique and White trap method proved useful in the recovery of nematodes from collected soil samples and infected cadaver, respectively. Molecular identification based on the amplification of the 18S rDNA and phylogenetic relationships revealed high affinity of the unknown EPN isolate 10 to Steinernema species and due to variation in evolutionary divergence distance, the unknown isolate was identified as Steinernema spp. isolate 10 . Isolates 35 and 42 revealed high similarity to Heterorhabditis zealandica strain Bartow (accession number: GU174009.1), Heterorhabditis zealandica strain NZH3 (accession number: EF530041.1) and the South African isolate Heterorhabditis zealandica strain SF41 (EU699436.1). Both Steinernema spp. isolate 10 and Heterorhabditis species could tolerate desiccation. Steinernema spp. isolate 10 was tolerant up to 11 days of desiccation exposure in loamy sand and up to 9 days of exposure in river sand, causing 26, 6% and 13, 4% cumulative larval mortality after 96 hours, post resuscitation by rehydration, respectively. Heterorhabditis spp. could tolerate desiccation up to 13 days of exposure and induced 26.6% cumulative larval mortality on both loamy and river sand after 96 hours post resuscitation. Swarming, aggregation, coiling and clumping behavioural characteristics were observed when Steinernema spp. isolate 10 was exposed to desiccation and Heterorhabditis species displayed no similar behavioural characteristics associated with desiccation tolerance. Morphological characteristics of the unknown Steinernema spp. isolate 10 have been described, and the thick cuticle and sheath which are both associated with tolerance to desiccation stress have been noted. The bacterial symbiont was isolated from larval hosts infected with Steinernema spp. isolate 10 and molecular identification through NCBI Blastn based on the 16S rDNA revealed high affinity to Xenorhabdus bacterial species. Phylogenetic relationships and evolutionary divergence estimates 16 revealed genetic variation and the species was identified as Xenorhabdus bacterial isolate. The genome assembly of Xenorhabdus bacterial isolate using CLC Bio revealed a total length of 4, 183, 779 bp with 231 contigs (>=400bp), GC content of 44.7% and N50 of 57,901 bp. Annotation of the assembled genome through NCBI PGAAP annotation pipeline revealed 3,950 genes (3,601 protein coding sequences (CDS) and 266 pseudogenes), 12 rRNAs and 70 tRNAs. RAST annotation revealed 55 of virulence, disease and defense subsystem features which are involved in the pathogenicity of Xenorhabdus bacterial isolate. The ability of EPNs to tolerate environmental stress is highly crucial and one of the determining factors for biocontrol potential and successful application, thus the indigenous desiccation tolerant EPN isolate, Steinernema spp. isolate 10 holds great potential as a biological control agent. The genome sequencing and annotation reveals insight to behavioural and physiological attributes of bacterial symbionts and this study will contribute to the understanding of pathogenicity and evolution of the bacteria–nematode complex. / GR 2016

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Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus. / Department of Biology

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Chrysopa carnea Stephens is widely distributed throughout the world. The larva is a potential control agent of pests in greenhouses, field crops and orchards. Its use as a biocontrol agent in integrated control is enhanced by its tolerance to some insecticides. Because of its considerable usefulness, studies to investigate the bionomics of this predator under both laboratory and field conditions were initiated. The study of population dynamics in alfalfa fields indicated that the major factors which govern the fluctuation of Chrysopa are: climate, food (e.g. aphids), diseases, parasites and agricultural practices (e.g. cutting of alfalfa hay). In each year and for each field, it was apparent that population levels of larvae rose to peaks five to six times; this suggests that there are five to six generations per year. The study of the effect of temperature and photoperiod on development in the laboratory showed that the egg, larva and pupa responded significantly to both temperature and photoperiod and an interaction between these two parameters was detected. Relative to fecundity and longevity, the investigations revealed that there was a photoperiod response only with respect to number of eggs laid and preoviposition period and that interaction between temperature and photoperiod was absent. In the case of the oviposition period, a response to temperature was detected and interaction between temperature and photoperiod existed. The different regimes had no significant effect on longevity of either sex. Consumption of prey differed significantly--being highest with beet armyworm eggs (Spodoptera exigua (Hubner) followed by corn earworm (Heliothis zea (Boddie) and tobacco budworm (Heliothis virescens (Fabricius). Fewer pea aphids (Acyrthosiphon pisum (Harris) were consumed than any of the lepidoterous eggs. Resistance to starvation differed significantly among the three instars; the newly-emerged Chyrsopa larvae had the shortest survival period. Studies on consumption of different instars of tobacco budworm by different instars of the predator indicated that the first instar was most vulnerable to attack by each of the first and third instars of the predator. Experiments were conducted to determine the effects of cold storage (4.4°C) of eggs and pupal cocoons for different periods of time. The results indicated that percent egg hatch and survival of emerging larvae were highest when stored for one week. Pupal storage for different periods had no detrimental effect on survival of pupae nor fecundity of emerging adults.

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