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No / The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.

Doctor of Philosophy / Department of Entomology / Kun Yan Zhu / Chitin metabolism represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely toned chitin synthesis and degradation and this process is absent in humans and other vertebrates. However, current understanding on this process and the involved enzymes is rather limited in insects. In this study, two chitin synthase genes (AgCHS1 and AgCHS2 or AgCHSA and AgCHSB), and 20 chitinase and chitinase-like genes (groups I-VIII) presumably encoding the enzymes for chitin biosynthesis and degradation, respectively, were identified and characterized in African malaria mosquito, Anopheles gambiae. Immunohistochemistry analysis and developmental stage- and tissue-dependent transcript profiling by using reverse transcription PCR, real-time quantitative PCR, and in situ hybridization revealed new information on these genes. Current understanding on chitin synthases is extended by the expression profiles such as the localization of AgCHS1 and AgCHS2 transcripts in eggs, AgCHS2 transcripts in the posterior larval midgut, AgCHS1 and AgCHS2 proteins in the compound eyes, and AgCHS2 enzyme in pupal inter-segments. Chitinase and chitinase-like genes are highly diverse in their gene structure, domain organization, and stage- and tissue-specific expression patterns. Most of these genes were expressed in several stages. However, some genes are stage- and tissue-specific such as AgCht8 mainly in pupal and adult stages, AgCht2 and AgCht12 specifically in foregut, AgCht13 exclusively in midgut. Functional analysis of each chitin synthase gene was conducted by using the chitosan/dsRNA nanoparticle-based RNA interference (RNAi) through larval feeding. The repression of the AgCHS1 transcripts which are predominantly expressed in carcass initiated from the mosquito larval feeding of dsRNA suggests the systemic nature of RNAi in mosquito larvae. In addition, silencing of AgCHS1 increased larval susceptibilities to diflubenzuron, whereas silencing of AgCHS2 enhanced the peritrophic matrix disruption and thus increased larval susceptibilities to calcofluor white or dithiothreitol. Furthermore, a non-radioactive method was adapted and optimized to examine the chitin synthase activity in mosquitoes. By using this method, diflubenzuron and nikkomycin Z show limited in vitro inhibition on chitin synthase at high concentration in cell free system, whereas no in vivo inhibition was observed.

This thesis describes the results of a three year study which collected larvae of the acorn barnacles Semibalanus balanoides on a rocky coastline in Fife, East Scotland. The nauplii larvae of S. balanoides are released from their parent in springtime in the United Kingdom and develop in the plankton for approximately one to two months. During this period they are transported some distance from the parent population and eventually return to the intertidal shoreline as a cyprid larval stage. The ‘decision’ to settle is a crucial point in the life history of the organism as most sessile organisms cannot move once this has occurred. The supply of larvae to intertidal shorelines was historically neglected until the 1980s. Patterns of settlement were largely considered irrelevant to distribution patterns of adults relative to postsettlement processes such as predation and competition. Despite the resurgence in “supply-side ecology” in the past 20 years there has been little development towards the measurement of larval supply. Consequently there has been very little description of fine scale or large spatio-temporal studies involving larval supply. This study demonstrates the first study directly addressing larval supply independently from larval settlement at mesoscales (metres to kilometres; days to years). Improvements were made to the passive larval trap described by Todd (2003). A conical opening was combined with the spiral trap design and a number of inlet areas were trialled (0.25cm², 0.5cm², 1cm² and 2cm²). These were tested across typical wave regimes measured with a new autonomous pressure sensing wave transducer and the 1cm² inlet was selected as the most appropriate trap design due to a balance between an increased per unit inlet larval capture and sufficient absolute larval capture so as to identify daily variation in larval supply to a site. Larval settlement has been studied extensively and is often used as a direct measure of rates of larval supply. It is widely assumed therefore that rates of settlement are a direct reflection of rates of supply, as long as settlement substrates and adult conspecific responses remain uniform. This thesis provides a means of accurately characterising the supply-settlement relationship for S. balanoides independent of substrate based responses and demonstrates that this is not the case. The relationship was found to be asymptotic, even at sites where there was low larval supply. It was concluded that density-dependent larva-larva interactions were present during the settlement of larvae and were relevant at daily temporal scales, limiting the rates of larval settlement proportional to larval density. There was no obvious effect on this relationship due to wave action however differences were observed between sites and years. Saturation of preferential environments within the tiles was observed resulting in a settlement preference cascade, with larvae being forced to settle in increasingly less preferable areas of the tiles with increasing larval density. Larva-larva interactions are demonstrated as having a considerable effect on the rates of settlement of S. balanoides.

The high-dose/refuge strategy is used globally to manage insect resistance development in genetically modified crops with insecticidal properties (Bt crops). The “refuge in a bag” (RIB) strategy is also being considered for deployment against several pest species. Busseola fusca, the target pest of Bt maize in South Africa, evolved resistance to Cry1Ab proteins. The objective of this study was to determine whether migrating B. fusca larvae are effectively controlled using the RIB strategy. A field study with a single-gene event (Cry1Ab) and a “pyramid” event (Cry1A.105 + Cry2Ab2) was conducted in which the migration patterns of B. fusca larvae in plots with different seed mixture treatments were studied. The experiment consisted of five seed mixture ratios (5%, 10%, 15%, 20% non-Bt seed and 100 % non-Bt seed as control). Natural infestation was augmented by artificial inoculation with neonate larvae into the central non-Bt maize plant of each plot. Rate of larval survival and migration, measured in terms of increase in number of plants per plot that exhibited borer damage was recorded at weekly intervals until flowering. A laboratory study was conducted to determine larval growth and survival when simulating migration between Bt and non-Bt maize plants. A feeding experiment in which larvae were reared on different types of maize (Bt and non-Bt) was conducted and larval survival and mass recorded after a 7-day feeding period. The incidence of damaged ears, stem damage and damaged internodes per stem were recorded and relationships between these variables determined by means of correlation analyses. A review was conducted in order to identify and discuss similarities and differences between the high-dose/refuge and seed mixture strategies. This was done to determine which strategy would be the most appropriate insect resistance management (IRM) strategy against B. fusca. The rate of survival and migration of B. fusca larvae was significantly higher in the plots with maize expressing Cry1Ab and control plots, than in plots with the pyramid Bt event. Older larvae exhibited improved growth and survival in the laboratory experiment when they were transferred from non-Bt to Bt plants. Positive correlations were found between early and late season damage, although some weaker than others. Plants of the “pyramid event” suffered less late-season damage than those of the single-gene event. Since the increase in number of damaged maize plants over time is associated with migration of older and larger larvae, the observed tendencies may indicate that the assumed high-dose does not kill larvae above a certain developmental stage. The high-dose refuge strategy seems to be the better option for delaying resistance development. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

La variabilité phénotypique a tendance à augmenter lorsque l'environnement est variable dans le temps ou l'espace. Cette thèse traite plus précisément de la variabilité des traits d'histoire de vie dans le cadre de populations fragmentées. En région Méditerranéenne, le Pélodyte ponctué, Pelodytes punctatus, se reproduit à la fois au printemps et en automne, en réponse aux variations temporelles de l'environnement. J'ai cherché à comprendre l'origine et les conséquences évolutives de ces stratégies de reproduction. Les deux périodes de reproduction produisent efficacement des descendants (plus en automne) et les têtards de printemps semblent souffrir de la compétition avec les têtards d'automne. Ces derniers se métamorphosent plus tôt et à une plus grande taille. Ces différences de développement ne s'expliquent pas par des différences génétiques entre des populations saisonnières. Au contraire, elles sont liées à la plasticité phénotypique et répondent à des conditions environnementales drastiquement différentes. Il semble plus favorable de se reproduire en automne. Le maintien des deux stratégies correspond à du bet-hedging ou un simple opportunisme. Par ailleurs, la fragmentation, phénomène croissant lié à l'anthropisation des milieux, diminue la taille des populations et augmente l'hétérogénéité génétique au sein d'une population. Il n'y a cependant pas de consanguinité au sein des populations étudiées mais une forte structuration en familles qui induit des associations allèles/phénotypes.Ce travail éclaire les variations de traits d'histoire de vie du Pélodyte et indique une grande plasticité phénotypique face aux variations de l'environnement. / Phenotypic variability tends to increase in temporally and spatially variable environments. This thesis deals with the variability of life-history traits in fragmented populations. In Mediterranean regions, Parsley frog, Pelodytes punctatus, breeds both in spring and in autumn, in response to temporal variations of its environment. I studied the origin and evolutionary consequences of its breeding strategies.Both breeding periods produce offspring (much more in autumn, though) and spring tadpoles suffer from intraspecific competition with older autumn tadpoles. Autumn laid juveniles are bigger and emerged sooner from the ponds. These developmental differences are not due to genetic differences between seasonal populations. They are explained by phenotypic plasticity in response to drastically different conditions. Even if it seems more favourable to breed in autumn, both strategies are maintained either by bet-hedging or pure opportunism.Besides, fragmentation, which increases with global changes, tends to reduce population effective size and increase genetic heterogeneity within populations. However, no inbreeding was found in the studied populations but a high family structure induced alleles/fitness correlations.Together, these results enlighten the variability of breeding strategies and larval traits in Parsley frog and indicate a high phenotypic plasticity in response to environmental variations.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Our research work is divided into three chapters. In the first chapter, synthesis of substituted phenolic compounds including halogenated di- and trihydroxybenzenes, aminophenols, and substituted di-tert-butylphenols, their redox potential, laccase oxidation, and mosquito anti-larval activities are discussed. The synthesized substituted phenols were found to be the substrates but not the inhibitors of laccase. An inverse correlation between the oxidation potential and the laccase oxidation efficiency of halogenated hydroxybenzenes and aminophenols was established. However, substituted di-tert-butylphenols were found to have anti-larval activities in mosquitoes resulting in the death of the larvae just before reaching pupation. Among the di-tert-butyl phenols studied, water insoluble, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (16), 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (14), and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (17) caused the mortility of 98%, 93%, and 92% of Anopheles gambiae larvae in the concentration of 182 nM, 3.4 µM, and 3.7 µM, respectively. In particular, compound 16 had similar anti-larval activities as compared to MON-0585, an anti-larval agent reported by Monsanto in the 70’s. In the second chapter, inhibition of protein kinase C (PKC) phosphorylation by substituted quinolines (PQs) is inverstigated. PQ compounds such as N-(3-aminopropyl)-6-methoxy-4-methyl-5-(3-(trifluormethyl)phenoxy)quinolin-8-amine (PQ1), N-(furan-2-ylmethyl)-6-methoxy-4-methyl)-5-(3-(trifluoromethyl)phenoxy)quinolin-8-amine (PQ11), and 6-methoxy-4-methyl-N-(quinolin-4-ylmethyl)-5-(3-(trifluoromethyl)phenoxy)quinolin-8-amine (PQ15) were found to inhibit PKC phosphorylation with IC50 values of 35 nM, 42.3 nM, and 216.3 nM respectively, among which PQ1 and PQ11 were found to be potent PKC inhibitors as comparable to that of staurosporine (IC50 = 33 nM). In chapter three, the tissue distribution of PQ1 and PQ11 in normal C57BL/6J mice and the effect of PQ1 on the normal tissues of mice were investigated. Substituted quinolines, PQ1 and PQ11 were distributed in the tissues in concentrations that were more than 40 folds of their effective dose. PQ1 and PQ11 were also found to penetrate the blood brain barrier and collect in the tissue in significant amounts. The administration of PQ1 and PQ11 had no effect in the normal behavior of the animals indicating no short term adverse effects. PQ1 was found to increase the expression of survivin, an anti-apoptotic factor and decrease the expression of cleaved caspase-3 and caspase-8, pro-apoptotic proteins. These studies suggests that PQ1 might have anti-apoptotic activities in normal cells, in contrast to the role of PQ1 in cancer cells where it has demonstrated to induce apoptosis. The study also indicated that PQ11 was better metabolized from the tissues over time as compared to PQ1.

The creation of the Mississippi River Gulf Outlet (MRGO) in 1963 by the U.S. Army Corps of Engineers (USACE) represents one of the most catastrophic, anthropogenic stressors ever to impact the Lake Pontchartrain estuary in southeastern Louisiana, USA. The artificial tidal pass provided a direct route from the Gulf of Mexico to New Orleans. It allowed for high saline waters to enter Lake Pontchartrain, resulting in detrimental changes to the biotic community of the Lake and surrounding wetland areas. In July 2009, the USACE closed the MRGO in hopes of restoring natural ecosystems. This study assesses changes in the adult, juvenile, and larval fish assemblages in the Lake from data taken before and after the closure. Water quality data were also examined for shifts related to the MRGO closure. Significant decreases in salinity were found following the closure, however no significant differences were found in adult or larval fish assemblages.

Karenia brevis, le dinoflagellé produisant des brevetoxines (PbTx), est la principale espèce d’efflorescences d’algues toxiques dans le Golfe du Mexique. Les effets de cette algue sur Mercenaria mercenaria et Crassostrea virginica sont méconnus tandis que les efflorescences coïncident avec la période de reproduction de ces espèces. Ce projet avait pour but de déterminer les effets i) d’une exposition à K. brevis en laboratoire et naturelle de terrain sur les processus physiologiques associés à la reproduction de M. mercenaria et C. virginica, et ii) d’une exposition à K.brevis sur la qualité et le développement des gamètes, embryons et larve de ces espèces. Suite à l'exposition des adultes de clams et d’huîtres à K. brevis, les paramètres physiologiques de la reproduction ont été affectés. La présence de PbTx dans les tissus des gamètes et le potentiel transfert maternel de PbTx à la progénie via les ovocytes, pourraient avoir entraîné les effets négatifs observés lors du développement larvaire.Les effets négatifs similaires causés par l'exposition des stades précoces à différentes préparations de cellules de K. brevis suggèrent que d'autres composés toxiques, en plus de PbTx, pourraient être impliqués dans la toxicité et, que la majorité des effets délétères se produisent durant les divisions embryonnaires.Le clam et l’huître américaine sont sensibles à K. brevis. Les effets négatifs sur les adultes et jeunes stades de vie, combinés à une exposition quasi-annuelle aux efflorescences de K. brevis, pourraient engendrer des perturbations majeures sur le recrutement des populations de ces espèces importantes, et avoir des répercussions environnementales et économiques. / The brevetoxin (PbTx) producing dinoflagellate, Karenia brevis is the most prevalent harmful algal bloom species in the Gulf of Mexico. The effects of this alga on Mercenaria mercenaria and Crassostrea virginica are poorly understood yet, blooms typically overlap with periods of reproduction and spawning in these species.The aims of this project were to determine the effects of i) laboratory and field exposure of K. brevis on the reproductive and related physiological processes of adult M. mercenaria and C. virginica and the quality of the offspring that were produced and ii) K. brevis exposure on gamete, embryo and larval development in these species.Following exposure of adult clams and oysters to K. brevis, negative effects were recorded on reproductive and physiological parameters. PbTx was recorded in gamete tissues, and maternal transfer of this PbTx to the offspring via the oocytes, may have resulted in the significant negative effects recorded on larval development up to the end of the lecithotrophic phase.The similar dose-dependent negative effects caused by direct exposure of gamete and early life stages to different cell preparations of K. brevis suggests that other toxic compounds in addition to PbTx may be involved in toxicity and, that the majority of negative effects occur during embryonic divisions.Hard clams and eastern oysters are susceptible to K. brevis exposure. The negative effects on adult and early life stages combined with the near- annual exposure to blooms of K. brevis could cause significant bottle-necks on the recruitment and population dynamics of these important species and, have wider reaching environmental and economic impacts.

Introdução: Assentamentos rurais são recorrentes arranjos sociais em expansão na Região Amazônica, que geram perturbações no ambiente natural e na respectiva ecologia de mosquitos vetores envolvidos na transmissão de malária. Objetivos: Elaborar revisão das espécies do gênero Anopheles que foram registradas na Região Amazônica. Para a pesquisa quantitativa, estabelecer potenciais fatores dos ambientes e das características das águas de diversos habitats larvais que possam estar associadas com a presença de espécies de anofelíneos, através de coletas em criadouros de diferentes graus de perturbação ambiental. Métodos: para a revisão das espécies que foram registradas na Amazônia, foram empregados termos de busca, utilizando sete bases bibliográficas de referência. Para os estudos em campo, foram realizadas capturas de larvas juntamente com estimação e mensuração de algumas condições dos habitats larvais, para comparação dos parâmetros analisados entre ambientes naturais e artificiais. Resultados: a revisão mostrou maior quantidade de artigos que notificaram a ocorrência do subgênero Nyssorhynchus, seguido pelos subgêneros: Anopheles, Stethomyia, Kerteszia e Lophopodomyia. O Anopheles darlingi se mostrou a espécie com maior ocorrência nos artigos levantados, muito pelo seu caráter epidemiológico. Importantes vetores auxiliares pertencentes ao subgênero 6 Nyssorhynchus também foram registrados em grande quantidade. Os resultados das análises conduzidas com os dados das capturas em campo demonstraram que as forças de transformação do ambiente natural favorecem o desenvolvimento de habitats larvais de espécies do subgênero Nyssorhynchus, incluindo o An. darlingi, além de um importante vetor secundário, o An. triannulatus. Discussão e Conclusão: projetos de assentamentos na Amazônia brasileira registram índices significativos de casos de malária, devido à ocupação de áreas naturais pelo homem. Este estudo mostrou que o subgênero Nyssorhynchus possui amplo registro na literatura de inquéritos entomológicos do gênero na floresta tropical equatorial, indicando que o agrupamento pode ser considerado endêmico às fronteiras de transformação na região, tendo assim importante papel na manutenção da transmissão da malária. Os estudos de campo apresentaram evidências de que formação de habitats artificiais favorecem formas iniciais de vida dos mosquitos com histórico vetorial para a região. Esses habitats larvais podem ser respostas ao uso do solo e das consequentes alterações provocadas no ambiente natural pelas atividades humanas. As modificações do ambiente natural selecionam e propiciam a formação de habitats para as espécies do Nyssorhynchus. Este agrupamento taxonômico engloba espécies que são consideradas vetores para a malária na região Neotropical. / Introduction: Rural settlements are recurrent social arrangements expanding in the Amazon region, which generate disturbances in the natural environment, affecting the mosquitoes ecology involved in malaria transmission. Objective: A systematic review of Anopheles species were recorded in the Amazon region. For the qualitative research, were establish potential factors of the environment and the characteristics of the waters of several larval habitats that may be associated with the presence of species of Anopheles mosquitoes, by surveys of the samples presents in larval habitats within environmental disturbance gradient. Methods: for the review of the species, were used search terms, in seven bibliographic databases of reference. For studies in the field, larvae were trapped along estimation and measurement of some conditions of the larval habitats, to compare the parameters analyzed between natural and artificial environments. Results: The review showed greater amount of articles that reported the occurrence of Nyssorhynchus subgenre, followed by: Anopheles, Stethomyia, Kerteszia and Lophopodomyia. The Anopheles darlingi proved the species with the highest occurrence in the papers showed, due to its epidemiological importance. Important auxiliary vectors belonging to the subgenus Nyssorhynchus were also recorded in large scale. The results of the qualitative shown that the natural 8 environment transformation forces favor the development of larval habitats of the species of subgenus Nyssorhynchus, including An. darlingi and an important secondary vector, An. Triannulatus. Discussion and Conclusion: settlement projects in the Brazilian Amazon recorded significant rates of malaria cases. This study showed that, the Amazon basin an important determinant of this phenomenon is the formation of artificial habitats that are occupied by early forms of life of mosquito vectors. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus. This taxonomic group includes species that are considered vectors for malaria in the Neotropics. Discussion and Conclusion: settlement projects in the brazilian Amazon recorded significant rates of malaria cases. This study showed that the review of the literature shows Nyssorhynchus subgenre ample record in entomological surveys in the equatorial rainforest, indicating that the group may be considered endemic to the borders of transformation in the region, and thus important role in the maintenance of malaria transmission. Field studies showed evidence that formation of artificial habitats favor early forms of the mosquito vector, with history for malaria in the region. These larval habitats may be answers to land use and consequent changes in the natural environment caused by human activities. Modifications to the natural environment select and provide the formation of habitats for species in Nyssorhynchus.