Published work submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the degree Doctor of Science in Medicine. / Andrew Chakane 2018

Francisella tularensis is a highly virulent Gram-negative bacterium causing the zoonotic disease tularemia. Arthropod-borne transmission plays an important role in transferring the disease to humans. F. tularensis induces very low amounts of pro-inflammatory cytokines during infection, due to inhibition of immune signaling pathways and an unusual structure of its lipopolysaccharide (LPS). To date, there is no vaccine available that is approved for public use, although an attenuated live vaccine strain (LVS) is commonly used as a model of the more infectious Francisella strains. To produce an effective vaccine it is important to understand the lifecycle of F. tularensis, including the interaction with the arthropod hosts. Drosophila melanogaster is a widely used model organism, which is increasingly being used in host-pathogen interaction studies as the immune pathways in flies are evolutionary conserved to the immune pathways in humans. An important part of the immune defense of D. melanogaster as well as of arthropods in general is the production of antimicrobial peptides. These peptides primarily target the bacterial membrane, inhibiting bacterial proliferation or directly killing the bacteria. The aim of this thesis was to establish D. melanogaster as a model for F. tularensis infection and as a model for arthropod vectors of F. tularensis. Also, to use D. melanogaster to further study the interaction between F. tularensis and arthropod vectors, with specific regard to the host immune signaling and arthropod antimicrobial peptides. F. tularensis LVS infects and kills D. melanogaster in a dose-dependent manner. During an infection, bacteria are found inside fly hemocytes, phagocytic blood cells, similar as in human infections. In mammals genes of the intracellular growth locus (igl) are important for virulence. In this work it is shown that the igl genes are also important for virulence in flies. These results demonstrate that D. melanogaster can be used as a model to study F. tularensis-host interactions. LVS induces a prolonged activation of several immune signaling pathways in the fly, but seem to interfere with the JNK signaling pathway, similarly as in mammals. Overexpression of the JNK pathway in flies has a protective effect on fly survival. Relish mutant flies, essentially lacking a production of antimicrobial peptides, succumb quickly to a F. tularensis infection, however, F. tularensis is relatively resistant to individual D. melanogaster antimicrobial peptides. Overexpressing antimicrobial peptide genes in wildtype flies has a protective effect on F. tularensis infection, suggesting that a combination of several antimicrobial peptides is necessary to control F. tularensis. The production of numerous antimicrobial peptides might be why D. melanogaster survives relatively long after infection. An intact structure of the lipid A and of the Kdo core of Francisella LPS is necessary for resistance to antimicrobial peptides and full virulence in flies. These results are similar to previous studies in mammals. In contrast to studies in mammals, genes affecting the O-antigen of F. tularensis LPS are not necessary for virulence in flies. In conclusion, this thesis work shows that D. melanogaster can be used as a model for studying F. tularensis-host interactions. LVS activates several immune pathways during infection, but interfere with the JNK pathway. Overexpressing the JNK pathway results in increased survival of flies infected with LVS. Despite rather high resistance to individual antimicrobial peptides, exposure to a combination of several D. melanogaster antimicrobial peptides reduces the virulence of F. tularensis.

Thesis (M. Sc.(Zoology and Entomology))--University of Pretoria, 2009. / Summary in English.

Historically, New Zealand was almost completely forested below the alpine treeline, but 1000 years of Polynesian and European colonisation has resulted in the destruction of nearly three-quarters of the original forest cover. I assessed historical patterns of deforestation and forest fragmentation in relation to all major topographical, climatic and anthropogenic variables that may drive forest loss. Much of the deforestation occurred in regions with drier climates, reflecting the fact that human population density has always been highest in areas with moderately dry climates and that dry forests burned much more readily and extensively. The large remaining tracts of forest are mainly restricted to high elevations, while the lowland forests have been fragmented into small, isolated remnants. Fragmentation of the surviving forests increases their susceptibility to edge effects and invasion by adventive species, indelibly altering the ecological communities they support. Although a large proportion of the remaining forest is owned or managed by the Department of Conservation, the distribution of that protection is greatly skewed towards areas of low economic value and is not representative of the relative conservation value of landscapes that differ in their environments and degree of forest cover. Forest cover in the majority of New Zealand landscapes has been reduced below the level of an expected extinction threshold of 30 % forest cover in the landscape, and ongoing deforestation threatens to force more landscapes below the critical threshold. Deforestation is still occurring across the country, and it is concerning that current deforestation rates in some areas are far greater than those observed in tropical, developing nations. I showed that the remaining forest fragments in New Zealand have complex, irregular shapes, and find ubiquitous evidence that core habitats within individual fragments are spatially discontinuous, comprising multiple, disjunct cores of small average area. Because population density of forest-interior species typically decreases with decreasing habitat area, multiple, disjunct cores support a lower total population size than a single, discrete core of the same total area. I found in a spatially explicit, landscape-level analysis of habitat fragmentation in New Zealand that simple core-area models consistently overestimate the carrying capacity of habitat fragments. Habitat fragmentation and habitat destruction are widely recognised as two of the leading threats to the continued maintenance of global biodiversity. The effects of habitat fragmentation on biodiversity fall into five categories that describe the spatial and landscape attributes of fragmented ecosystems; (1) fragment area, (2) edge effects, (3) fragment shape, (4) fragment isolation, and (5) matrix structure. Each attribute affects species individually according to their particular biological requirements and life history strategies, leading to complex, and often conflicting, sets of results in the empirical literature. Furthermore, it is now apparent that the effects of fragmentation can take many decades to become apparent and that the spatial arrangement of habitat fragments can interact with other ecological processes to magnify the detrimental impacts of fragmentation on species. I synthesised the published effects of habitat fragmentation on the morphology, distribution and abundance of invertebrate populations, species and communities, and present examples of time lags and synergies from the fragmentation literature. I explicitly considered the underlying mechanisms determining the responses ofindividuals to fragmentation and discussed the role of species traits in determining species vulnerability to changes in the spatial attributes of fragmented landscapes. I sampled 35,461 beetles from a fragmented forest and matrix system in New Zealand over very large gradients of fragment area (10-2 to 106 ha) and edge distances (up to 1,024 m from the forest edge into both the forest and the adjacent matrix interiors). The beetle fauna was very diverse, with 893 species identified in 65 families, representing nearly 20 % of the known species in New Zealand. Beetle communities were strongly structured by forest fragmentation, but in species-specific ways. Distance to edge was consistently shown to have the largest effect on community composition, but, surprisingly, an interaction between area and distance to edge had a stronger impact on community structure than fragment area alone. I developed a new method to partition the variance in community composition that was explained by putative area and edge effects. The method uses backwards stepwise regression to determine significant predictors of gradients in beetle species composition that were identified by canonical ordination. I found that edge effects were driven partially by small-scale alterations to microhabitat and microclimate and partially by changes in landscape composition that varied with distance to edge. In contrast, fragment area effects were driven primarily by edge effects, the strength of which varied significantly with fragment area. I took a novel approach to characterising the responses of 185 common species to habitat edges by modelling species abundances across edges with a general logistic model that described sigmoid trends in abundance for forest specialist and matrix specialist species, as well as unimodal trends in abundance for edge specialist species. I used the second derivatives of the logistic and unimodal models to statistically determine the width of species response zones to edge effects. Beetle species responses to forest edges occurred over far greater scales than previously suspected, with edge response zones for some species extending for more than 1 km. Average edge response zones were 194 m wide and, for many species, began in the forest but extended into the adjacent matrix. Species were categorised according to their responses to fragment area and distance to edge. Closely related species were expected to be placed in similar response categories because they are predicted to share suites of traits that determine their susceptibility or resilience to fragmentation by virtue of common ancestry. Despite many species exhibiting responses that could be grouped into categories, individual species responses to fragmentation were largely idiosyncratic with even closely related species exhibiting strongly contrasting responses to fragmentation.

Thesis (Ph. D.)--Faculty of Rural Management, University of Sydney, 2004. / Bibliography: leaves 127-147.

Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 5 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Rural Management. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [177]-194).

The chelicerates (e.g., spiders, mites, scorpions, harvestmen and horseshoe crabs) are oneof the oldest arthropod clades, arising in the Cambrian. The chelicerates display a conserved body plan comprising of an anterior prosoma and a posterior opisthosoma. The prosoma comprises the segments bearing the chelicerae, pedipalps and the four pairs of walking legs. The opisthosoma is more variable, and either contains no appendages (e.g., in harvestmen), or extremely derived appendages (e.g., the spinnerets of spiders and the pectines of scorpions). Recent evolutionary developmental (evo devo) studies on arachnids and other arthropods have shown that a conserved suite of developmental genes pattern the chelicerate body plan, with slight modifications in their expression patterns correlating with morphological diversification and evolution of segments and the appendages that they bear. Although a few tractable evo devo chelicerate models are currently in use, there is a noted paucity of evo devo studies on mites, let alone embryological studies on mites using modern microscopic techniques. The following dissertation aimed at filling the gap in the current knowledge of mite evolution and development by studying the development of the emerging mite model mite Archegozetes longisetosus. Four main aims that focused on answering questions surrounding mite and chelicerate evolution were the subjects of the dissertation research. These aims included a.) determining the segmental composition of the mite opisthosoma, b.) determining how the segment of the suppressed fourth pair of walking legs is formed during embryogenesis, c.) determining how can the appendage-patterning genes, i.e., the "limb gap genes," aide in elucidating arthropod phylogeny and d.) determining how the evolutionarily conserved Hox genes pattern the reduced mite opisthosoma. The findings from this project include that the acariform mite opisthosoma has been reduced to two terminal segments, that the formation of the segments bearing the fourth pair of walking legs is tied to opisthosomal segmentation in A. longisetosus, that mites retain chelicerae comprised of three segments, that only the proximal elements of the fourth pair of walking legs are patterned during development in A. longisetosus, and that the mite homologues of the Hox genes Ultrabithorax and Abdominal-B are only expressed in a single segment in the opisthosoma in A. longisetosus. These findings are placed within a comparative framework to aid in the understanding of the evolution of arthropod and chelicerate body plans.

Forests provide humans with a range of valuable ecosystem services, but current forest management practices may increase susceptibility to damage from disturbances such as pest insects. One such practise is the harvesting of residue, which decreases the amount of dead wood in the forest. Dead wood has been shown to be of importance for biodiversity and could decrease vulnerability to biotic disturbances. Sustainable forest management and conservation of biological diversity is required to maintain ecologically healthy forests. I aimed to investigate if biodiversity connected to dead wood could contribute to insect pest mitigation. I sampled ground-dwelling arthropods with pitfall traps in monoculture pine stands in Central Sweden on plots where dead wood had been either added or removed. To relate catches from pitfall traps to predation pressure of pest insects, I used plasticine model larvae to measure the attack rate. Contrary to my expectations, total arthropod abundance was higher when dead wood had been removed. Furthermore, the presence of dead wood had no effect on total arthropod diversity and diversity and abundance of predators. There was no relationship between predator catches and attack rates on plasticine model larvae. My results contradict previous findings that dead wood promotes biodiversity and pest mitigation. This highlights the need for studies targeting the more specific effects of different types of dead wood, in different types of forest stands on specific taxonomic groups and ecological processes, as results do not appear to be uniform across studies.

Various models of density dependence predicted different evolutionary outcomes for Helicoverpa zea, Diabrotica virgifera, and Ostrinia nubilalis using simple and complex resistance evolution models, different dose assumptions and refuge proportions. Increasing available refuge increased durabilities of pyramided Plant-Incorporated-Protectants (PIPs), especially between 1-5%. For some models of density dependence and pests, additional refuge resulted in faster adaptation rates. Significant considerations should be given to a pest’s intra-specific competition in simple and complex theoretical models when designing insect resistance management plans. Life-history, refuge, and dose characteristics of a PIP had different effects on the adaptation rate of a generic pest of Bt, and unexpected outcomes occurred. Intrinsic growth rate ‘R0’ was the strongest evolutionary force, and large R0’s reduced time to resistance for a high dose PIP to similar levels as projected for a low dose PIP. This was caused by differential density dependent effects in refuge and Bt fields that elevated generational resistance increases beyond those from selection alone. Interactions between density dependence and R0 were always present and further affected the life-time of the PIPs. Varying ‘average dispersal distance’ did not affect evolutionary outcomes; however, increasing the proportion of the population engaging in dispersal often increased the durability of high dose PIPs. When resistance genes spread from a hypothetical hotspot, local resistance phenomena developed in the immediate surroundings. Higher growth rates lead resistance to spread faster through the landscape than lower rates. Increasing available refuges slowed adaptation rates to single PIPs and low dose pyramids, although non-linear trends were possible. Integrated Pest Management (IPM) practices at the onset of PIP commercialization slowed pest adaptation rates. For corn rootworm, interspersing non-selective periods with IPM+IRM delayed resistance evolution, yet crop rotation was the best strategy to delay resistance. For bollworm inclusion of isoline corn as an IPM tool did not increase the life-time of the PIP. A local resistance phenomenon for rootworm was maintained immediately surrounding the hotspot; random selection of mitigatory strategies in the landscape slowed adaptation rates while mitigation in the hotspot alone did not. Mitigation extended the life-time of the pyramid minimally for both corn rootworm and bollworm.