Os inibidores inibidores de serino proteinases do tipo Bowman-Birk (BBI) possuem dois sítios ativos e são encontrados em plantas das famílias Fabaceae e Poaceae. Neste trabalho foi apresentada a estrutura primária e o padrão de expressão de 14 seqüências expressas (EST, expressed sequence tags) de BBI putativos encontradas no banco de dados do "Projeto Transcriptoma da Cana-de-açúcar" (SUCEST). Estas quatorze seqüências foram utilizadas em conjunto com outras 87 seqüências de BBI previamente descritas e depositadas no banco de dados "GenBank" para a construção de árvores filogenéticas da família BBI. A análise filogenética mostrou que os BBI de monocotiledôneas e dicotiledôneas podem ser claramente separados em diferentes grupos e a topologia das árvores filogenéticas sugere um padrão evolutivo diferente das famílias de BBI em plantas. Os inibidores de dicotiledôneas são bem conservados e acumularam diferenças sutis durante a evolução. Em contrapartida, os inibidores de monocotiledôneas são altamente variáveis, indicando a ocorrência de um processo evolutivo interessante, baseado em eventos de duplicação intragênica e mutação. Dois inibidores de serino proteinases, um de tripsina e outro de quimotripsina, derivados do gene que codifica o inibidor Bowman-Birk de soja, foram expressos na superfície do fago filamentoso M13 e utilizados para a construção de bibliotecas de variantes. Para tal foram feitas mutações em quatro aminoácidos do sítio ativo destes inibidores e duas bibliotecas de expressão na superfície de fagos filamentosos foram construídas. Posteriormente, estas bibliotecas foram utilizadas para a seleção de variantes que melhor interagiam com a tripsina bovina e de Diatraea saccharalis e com as enzimas digestivas presentes no extrato intestinal desta praga. Os variantes selecionados foram seqüenciados, analisados e caracterizados. Os resultados mostraram que a técnica de expressão na superfície de fagos filamentosos foi eficiente para selecionar novos inibidores. Além disto, com as mutações realizadas, foi possível transformar a alça de inibição de quimotripsina em uma alça de inibição de tripsina. / The Bowman-Birk inhibitors (BBIs) are double headed inhibitors of serine proteinase found in plants from Fabaceae and Poaceae families. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag (SUCEST) database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the "GenBank" database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events. Two serine-type proteinase inhibitors, a trypsin and a chymotrypsin, both derived from the soybean Bowman-Birk inhibitor, were expressed on the surface of a filamentous phage. Site mutations were made in four positions of the reactive sites of these inhibitors and two phage-display libraries were constructed. Later, these libraries were used to select better ligands to the bovine and Diatraea saccharalis trypsin and to the midgut enzymes of this insect pest. The selected variants were sequenced, analyzed and characterized. The results showed that the phage-display technique is efficient to select new proteinase inhibitors. Furthermore, it was possible to modify the chymotrypsin loop into a trypsin loop using the library constructed by the insertion of a degenerated primer.

The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.

Anredera cordifolia (Basellaceae), Madeira vine, is a perennial, semi- succulent climber native from Paraguay to southern Brazil and northern Argentina. It has a history of weediness and difficulty of control once established. In South Africa Madeira vine has a wide range and distribution with altitudes ranging from 10-1800m above sea level. Described as a transformer species, its sheer weight is capable of breaking branches off trees, causing the potential collapse of forest canopies. Chemical and mechanical control methods are expensive, labour intensive and may provide only temporary relief. A biological control programme was therefore initiated in 2003. Cf Phenrica sp. 2 (Coleoptera: Chrysomelidae: Alticinae), was field collected from A. cordifolia in Brazil, SSW of Cascavel in the Paraná Province during a survey in November 2003. Eggs are laid in groups of 16 with the average fertility rate being 89%. After going though three larval instars, the larvae pupate in the soil with the adults eclosing after a period of 17 days. The total developmental time for a generation from egg to egg ranges between 7-8 weeks. Biological traits that favour the flea beetle as a possible biological control agent include long-lived adults (up to 5 months) and multiple generations during the summer period. Both adults and larvae feed extensively on leaves and stems and although developmental rates will slow down during the winter period, no indication of a definite diapause was found under the prevailing laboratory conditions. After completing the larval no-choice trials with twenty-six plant species from 14 plant families Phenrica sp. 2 proved to be adequately host specific, as larval development was only supported by 3 Basellaceae species (including the control A. cordifolia) and one Portulacaceae species. All of these are introduced species in South Africa. The only indigenous Basella species could not be tested as it has a very marginal distribution, and because it’s inconspicuous nature, it is seldom seen or collected. Adult multi-choice trials were restricted to species that could sustain larval development to give some indication of the acceptability of these species for adult feeding and oviposition. Although adult feeding was initially concentrated on B. alba, the oviposition preference was clear-cut as females only oviposited on A. cordifolia. In order to quantify the impact of Phenrica sp. 2 on plant biomass and to assess the incidence and intensity of foliar damage, a pair of adults was confined to the host plant, for 2 generations, with different levels of larval densities. The results indicated that the host plant, due to both larval and adult feeding, suffered leaf losses of up to 55%. Anredera cordifolia was however still capable of enlarging the root mass despite suffering huge leaf losses. This would imply that A. cordifolia has an effective re-growth capacity and it will only be vulnerable to attack of the storage organs that enable re-growth, or to repeated attack of other plant parts through which reserves are exhausted. Unfortunately the period of exposure (24 days) was too short to prove that Phenrica sp. 2 impacts on the below ground dry mass, but should the plant be completely defoliated, as was observed in the field, the host plant would be forced to deplete stored resources. Phenrica sp.2 has shown to be very host specific and although A.cordifoia loses its leaves during the winter period in most provinces in South Africa, the adults are long-lived and should be able to survive the leafless periods. Further more the relatively short life cycle, high fecundity and 3 generations per year should theoretically insure a strong population build-up that would improve the chances of establishment in the field. All indications are that Phenrica sp. 2 is an agent well worth considering for the biological control of A. cordifolia.

Varieties of Lantana camara (lantana) have been introduced into many countries of the world as ornamental plants and have become invasive weeds in many countries including South Africa. In South Africa, it mostly invades the sub-tropical eastern and northern range. Mechanical and chemical control options are expensive and ineffective. A biocontrol programme was initiated in South Africa in 1961. To date, 22 insect species, and a fungus have been introduced, of these 10, and the fungus have established. Three indigenous lepidopteran species and an exotic generalist pest mealybug are also associated with the weed. The variable success of some of the agents released on L. camara worldwide has been ascribed to a few factors. One important aspect is the large range of varieties encountered in the field. It is therefore essential to be able to predict the possible establishment and impact of agents on many varieties. Laboratory trials on five of the established agents showed clear varietal preferences. In the field, most of the biocontrol agents had limited geographic ranges, linked to altitudinal conditions, as higher populations were recorded at low lying warm summer rainfall areas. A pink and orange flower corolla lobe and throat colour combination and plants with few to medium leaf hairs were most abundant in South Africa. Most of the agent species had individual preferences towards different flower colour combinations, as the agents built up different population levels on varieties in the field, within the suitable geographic region for the insect species. Eight agents preferred smooth leaved varieties, while three preferred hairy leaves, and three had no specific preference to leaf hairiness. Varietal preferences thus did play a significant role in agent populations and accompanied impact achieved in the field. New candidate agents need to be proven specific under quarantine conditions and the results extrapolated to predict specificity in the field, while avoiding potential non-target effects. Many authors have questioned the validity of laboratory host specificity trials. The conventional wisdom is that insects portray a far wider host range in the laboratory than what they would do in the field. In other words, laboratory studies measure the physiological host range of an agent and are conservative and usually don’t reflect the ecological host range of agents in the field. To avoid unnecessary rejections of biocontrol agents, this study has made a retrospective study of the host specificity of agents established in the field. Their laboratory and field host ranges were compared and it was found that virtually all the agents reflect similar or less non-target effects in the field than predicted during multiple choice trials. Of the 14 agents, only one introduced species, Teleonemia scrupulosa, and the indigenous species, Hypena laceratalis and Aristea onychote were able to sustain populations on non-target species in the field in the absence of L. camara. Insect populations on non-target species were much reduced compared to that on L. camara. Furthermore non-target effects were only recorded on plant species closely related to the target weed. The multiple choice trials therefore predict field non-target effects accurately. Predictions of non-target effects of candidate agents can therefore be accurately predicted by laboratory studies, in terms of species likely to be affected and to what extent. One field that need further study though is the impact of non-target effects, especially on Lippia species by L. camara biocontrol agents.

Augmenter la diversité végétale au sein même du champ permet de réguler les populations de ravageurs dans de nombreux agroécosystèmes. Les mélanges variétaux (diversité intraspécifique) ou les associations de cultures avec une plante compagne (diversité interspécifique) sont considérées comme des pratiques agroécologiques prometteuses pour les systèmes de culture à bas intrants ou l'agriculture biologique. En effet, ces pratiques favorisent de nombreux services écosystémiques tels que la régulation des ravageurs, des maladies ou des adventices, ainsi que la fertilisation azotée. Cependant, le potentiel de régulation des ravageurs du blé par la combinaison de ces deux pratiques de diversification n'a pas encore été étudié.Nous avons combiné ces deux pratiques dans le cadre d'expérimentations menées en plein champ et sur deux saisons de culture, afin d'examiner leurs impacts sur les populations de pucerons et d'ennemis naturels. Nous avons également évalué le potentiel de régulation des ravageurs en mesurant les taux de prédation de proies sentinelles.La combinaison des diversités intra- et interspécifique n'est pas plus performante pour réduire les populations de pucerons que les pratiques prises séparément. L'association de culture blé-trèfle tend à être moins infestée par les pucerons, tandis que le mélange variétal est plus infesté que la variété la moins sensible. Les variations annuelles des conditions climatiques impactent fortement le développement du blé et du trèfle, ainsi que la date d'apparition du pic de puceron. Le rendement du blé, ainsi que le taux d'azote du grain sont réduits par l'association de culture par 7 à 10%, mais pas par le mélange variétal. La présence d'un couvert de trèfle dans les champs de blé, semble avoir favorisé la biodiversité fonctionnelle, particulièrement les ennemis naturels tels que les carabes, mais pas le mélange variétal. Les résultats sont variables selon la famille d'arthropodes concernée et leur position au sein du couvert végétal (au sol ou dans le feuillage). Le couvert de trèfle et le champ ont influencé la composition de la communauté de carabes prédateurs. Les taux de prédation des proies sentinelles n'ont pas été impactés par les pratiques de diversifications.En laboratoire, nous avons évalué comment l'association du blé avec des légumineuses (trèfle ou pois) pouvait modifier le comportement du puceron du blé Sitobion avenae en terme de location de sa plante hôte et du développement de la population. Les pucerons ont résidé moins de temps sur le blé quand il était associé à du trèfle. Les populations de pucerons se sont moins développées dans les associations du blé avec une légumineuse par rapport à du blé seul, mais si l'on prend en compte la biomasse du blé, seulement l'association blé-trèfle a considérablement réduit les densités de pucerons sur le blé. Ainsi l'espèce associée et sa densité sont des paramètres importants qui devraient être pris en compte dans les études sur la diversité interspécifique, car ils pourraient expliquer la grande variation dans les résultats rapportés par les analyses bibliographiques.Nos résultats suggèrent qu'augmenter la diversité cultivée au sein du champ peut aider à réguler les pucerons dans une certaine mesure, mais la combinaison des deux pratiques de diversification ne résultent pas en un trade-off entre la régulation des ravageurs et les performances agronomiques particulièrement attractifs pour les agriculteurs. / Increasing intrafield plant diversity has been shown to regulate pest populations in various agroecosystems. Polyvarietal mixtures of a crop species (intraspecific diversity) or associations of a crop and a companion plant (interspecific diversity) are both considered as promising agroecological practices for low-input or organic agriculture systems by providing several ecosystem services such as pest, disease and weed control, and nitrogen fertilization. However, combining both diversification practices has not been studied yet in perspective of winter wheat pest control.In organic field experiments over two growing seasons, we combined both practices and examined the direct impact on aphid and natural enemy populations and on wheat production. We also investigated the potential pest regulation service through the assessment of the rate of predation by using sentinel preys.Results show that combining intra- and interspecific diversity did not outperform each practice individually in reducing aphid populations, thus not clearly showing synergetic effects. Taken separately, intercropping tended to have lower aphid infestation, while it the cultivar mixtures was more infested by aphids than the least susceptible cultivar. Yearly variation in climatic conditions strongly impacted wheat and clover development, as well as the appearance of aphid peaks. Wheat yields and grain nitrogen content were reduced in intercropping by 7 to 10%, but not in cultivar mixtures. Functional biodiversity, especially natural enemies such as ground beetles, tended to be positively correlated to the presence of a clover cover in the wheat fields (interspecific diversification), but did not respond to the wheat cultivar mixture (intraspecific diversification). Results varied according to the family of arthropods concerned and their position within the vegetation layer (ground dwelling or foliage dwelling arthropods). The cover of white clover and the field context influenced the community composition of predatory ground dwelling beetles. Rates of predation on sentinel preys were not influenced by any of the diversification practices.Under laboratory conditions, we evaluated how combining wheat and legumes (clover or pea) modifies the behaviour of the cereal aphid Sitobion avenae in terms of host-plant location, and population growth. We observed that aphids’ residence time on wheat was decreased when this host-plant was intercropped with clover. At the population level, wheat-legume intercrops reduced the number of aphids on wheat plants compared to wheat sole crops but if we take into account plant biomass, only intercropping clover with wheat significantly reduced aphid densities on wheat. The species used as non-host plants and their density are important parameters that should be taken into account in studies on intercropping systems and that may explain the large variability in the results observed in the literature.Our findings suggest that intrafield diversification may regulate wheat aphids to some extent, but combining the two diversification practices did not result in an interesting trade-off between pest regulation and wheat production in real farming conditions.

The two-spotted spider mite (TSM), Tetranychus urticae Koch, is an important pest of many greenhouse and field crops worldwide. The development of resistance in TSM populations to chemical acaricides, allied with public health concerns about pesticide residues, has motivated the search for alternative control measures to suppress the pest. Hyphomycetous fungi are promising agents for mite control and the fungus Beauveria bassiana (Bb) (Balsamo) Vuillemin was investigated in this study as a biocontrol agent. The principal objectives of this study comprised: a) screening Bb strains for their pathogenicity against T. urticae; b) testing the effect of adjuvants on the efficacy of Bb; c) studying the effect of plant type on persistence of Bb and the efficacy of control of Bb against T. urticae; d) evaluating the field efficacy of Bb applications against T. urticae; e) testing the compatibility of Bb with selected fungicides; and f) assessing the synergy between Bb and soluble silicon for T. urticae control. Screening bioassays of sixty-two strains of Bb identified the two most effective strains, PPRI 7315 (R289) and PPRI 7861 (R444), that caused mortality levels of more than 80% of adult mites at 9 d post-inoculation with 2 × 108 conidia ml-1. These strains performed significantly better than the Bb commercial strain PPRI 5339, in laboratory bioassays. The two strains also attacked mite eggs, causing 53.4% and 55.5% reduction in egg hatchability at 2 × 108 conidia ml-1 respectively. However, PPRI 7861 showed relatively higher production of conidia in culture and was, therefore, selected for further trials under greenhouse and field conditions. Greenhouse evaluations of the effects of two adjuvants (Break-thru® and a paraffin oil-based emulsion) on efficacy of Bb demonstrated a higher efficacy of the biocontrol agent (BCA) when it was applied with Break-thru® or the oil solution than with water alone. Moreover, Bb conidia applied in Break-thru® solution resulted in greater control of TSM than conidia applied in the mineral oil. There was also a dose-response effect and the control of TSM by Bb increased when the concentration of conidia was increased. The control of TSM by Bb in beans (Phaseolus vulgaris L), cucumber (Cucumis sativus L.), eggplant (Solanum melongena L.), maize (Zea mays L.) and tomato (Solanum lycopersicum L.) was tested in greenhouse trials. On these crops, the persistence of conidia declined over time. The rate of decline was significantly higher on maize. However, TSM mortality was positively correlated with the amount of conidia deposited on leaves immediately after spraying, rather than their persistence over time. Higher levels of mortality of TSM due to Bb application were observed on beans, cucumber and eggplants, suggesting that the type of crop must be taken into consideration when Bb is applied as a BCA. Field efficacy of Bb against mites was evaluated in two trials on eggplants. Based on assessment of population densities of mites and leaf damage assessments; both trials showed that the strain PPRI 7861 controlled TSM in the field. Two commonly used fungicides, azoxystrobin and flutriafol, were investigated in vitro tests on culture medium and laboratory bioassays on detached bean leaves (Phaseolus vulgaris L.) for their effects on Bb. Azoxystrobin (a strobilurin) was less harmful to Bb while flutriafol was found to be inhibitory. Another important finding of this study was the substantial enhancement of Bb efficacy by soluble silicon. When Bb was combined with soluble Si, the control of TSM was better than when either of the two products was applied alone. Moreover, application of soluble Si as a plant fertilizer in hydroponic water nutrient increased accumulation of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase enzymes in leaves of plants infested with TSM. Increased activity of these defense enzymes in leaves deters feeding behaviour of mites. We suggested that feeding stress renders them susceptible to Bb infection, which would explain the synergy observed between the two agents. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Thesis (M. Tech) --Central University of Technology, Free State, 2008 / Aphids (Hemiptera: Aphididae) are amongst the most destructive insects in agricultural crop production systems. This reputation stems from their complex life cycles which are mostly linked to a parthenogenetic mode of reproduction, allowing them to reach immense population sizes within a short period of time. They are also notorious as important and efficient vectors of several plant viral diseases. Their short fecund life cycles allow them to be pests on crops with a short growth period, e.g. lettuce (Lactuca sativa L.). It is common practice to provide this crop with some degree of protection from environmental extremes on the South African Highveld. Shadehouses are popular in this regard, but aphids are small enough to find their way into these structures, and their presence on lettuce is discouraged due to phytosanitary issues. In addition, the excessive use of insecticides is criticized due to the negative influence on human health, and because aphids can rapidly develop resistance. This necessitates the use of alternative control options in order to suppress aphid numbers. Biological control is popular in this regard and the use of predatory ladybirds (Coleoptera: Coccinellidae) is a popular choice. This study investigated the aphid and coccinellid species complex encountered under varying shadehouse conditions on cultivated head lettuce in the central Free State Province (South Africa). Their seasonality was also examined, along with variations in their population size throughout a one-year period. Finally, the impact of varying aphid populations on some physical characteristics of head lettuce was examined, and recommendations for aphid control (using naturally occurring coccinellid predators) were made. Two shadehouse structures were evaluated during this study. One was fully covered with shade netting and designed to exclude the pugnacious ant, Anoplolepis custodiens (Hymenoptera: Formicidae), while the other was partially covered with shade netting (on the roof area) allowing access to the ants. Six cycles of head lettuce were planted and sampled four times during each cycle. These were scheduled to monitor the seedling, vegetative and heading stage of lettuce. Four important aphid species were recorded on the lettuce, namely Acyrthosiphon lactucae, Nasonovia ribisnigri, Myzus persicae and Macrosiphum euphorbiae. Both structures harboured similar aphid and coccinellid species, but their population dynamics differed. A. lactucae dominated in the absence of A. custodiens in the fully covered structure (whole study), while N. ribisnigri dominated in the partially covered structure in the presence of these ants during the warmer months (December – January). M. euphorbiae replaced this species as the dominant species in the absence of A. custodiens (April – September). M. persicae occured during the winter (May – August) in the fully covered structure. Promising coccinellid predators were Hippodamia variegata and Scymnus sp. 1, and to a lesser extent, Exochomus flavipes and Cheilomenes lunata. However, the fully covered structure hampered the entrance of the larger adult coccinellid species, resulting in their lower occurrence. Aphid and coccinellid activity peaked during the summer months (October – January), and the fully covered structure attained the highest aphid infestation levels and coccinellid larval numbers during this time. On the other hand, aphid numbers were higher in the partially covered structure during the cooler months of the year (April – July) and this structure also harboured more adult coccinellids. In most cases, aphid infestation levels did not affect the amount of leaves formed. However, symptomatic damage in terms of head weight reduction did occur under severe infestation levels. Specific environmental conditions within a shadehouse structure concurrently contributed to this reduction, with less favourable conditions accelerating this condition. Results from this study have shown that even though the type of shadehouse structure does not influence the insect species complex found on lettuce, it does have an influence on detrimental and beneficial insect population dynamics. Aphid species infesting lettuce have been identified, along with coccinellid predators that could potentially be used in their control. Both types of structures had advantages and disadvantages, and therefore, decisions concerning shadehouses should not be focused on which type of structure to use, but rather which type of structure to use during different seasons of the year.

No description available.

Interactions between insects have been shown to be important regulators of population abundances and dynamics as well as drivers of spatial segregation and distribution. These are important aspects of the ecology of insects used in biological control and may have implications for the overall success of a particular programme. In the history of biological control there has been a tendency to release a suite of agents against a weed, which in some cases has increased the level of success, while in others little change has been observed. In most of these cases the implications of increasing the level of complexity of the system is not taken into account and there is little research on the effect of releasing another agent into the system. A brief meta-analysis was done on all the biological control programmes initiated in South Africa. Emphasis was placed on multi-species releases and the effects that overlapping niches were having on the number of agents responsible for the success of a programme. Where overlapping niches were present among agents released the number of agents responsible for success was lower than the number established. Water hyacinth, Eichhornia crassipes (Martius) Solms-Laubach in South Africa has more arthropod agents released against it than anywhere else in the world, yet control has been variable. If the biology and host utilisation of all the agents against water hyacinth is considered, a definite overlap of niches is apparent in at least one life stage of all the agents. Therefore the probability of these insects interacting is high, especially if they are established at the same site in the field. Three of the insects released in South Africa have been selected to investigate possible interactions. They are Neochetina eichhorniae Warner, Neochetina bruchi Hustache and Eccritotarsus catarinensis (Carvalho). Y-tube olfactometer bioassays were used to measure responses of these insects to water hyacinth with prior feeding damage by either conspecifics or heterospecifics. This was done to determine whether olfactory cues played a role in host acceptability and avoidance of conspecifics or heterospecifics. The insects were given a choice between damaged and undamaged plants in various combinations. There was a significant preference for the undamaged plants when given a choice between undamaged and damaged plants. However when the insects were given a choice between two damaged plants there was no discrimination between heterospecific or conspecific damaged plants. This may indicate that there is little or no ecological cost for the insect to share a plant with other insects utilising a similar resource. Insect – insect interactions were investigated in a common garden plot experiment to measure the impact that pairwise combinations of the insect may have on their performance. There was a significant interaction between the mirid E. catarinensis and the weevil N. eichhorniae, with the weevil not performing as well when in combination with the mirid than when alone. Interestingly there was a negative interaction between the two weevil species when in combination, however it was impossible to determine which species was being affected if not both. None of the insects performed significantly better when in combination with another insect. A field study on Wriggleswade Dam in the Eastern Cape, South Africa was initiated to determine whether the relationship between the mirid E. catarinensis and the weevil N. eichhorniae could be determined in the field. The performance of the insects at the different sites in the field suggests that there was an interaction between the agents. This interaction did not limit the establishment of either insect at a site, but it did result in one insect dominating at a site over another. Interactions between the three species of insect tested in this thesis suggest that there are both negative and neutral relationships between them. A basic comparison between the insect performances from 15 sites around the country was done to determine if the spatial segregation observed in the field could be extrapolated to the natural South African situation. The interaction observed between N. eichhorniae and E. catarinensis does seem to extrapolate to the general South African situation where there is definite spatial segregation on a landscape level. The co–occurrence of the two Neochetina weevils at these sites suggests that the negative relationship observed between them in the common garden experiment does not extrapolate to the field. The results from this thesis suggest that the interactions between the agents tested would not limit establishment or have significant ramifications on performance. However, there may be spatial and temporal segregation of these species in the introduced range.

O uso de pesticidas constitui um importante aspecto na agricultura moderna, com inquestionável beneficio na produção agrícola. Porém a contaminação dos alimentos por pesticidas constitui um sério risco a saúde do consumidor. A determinação de resíduo de pesticidas em alimentos envolve procedimentos laboriosos, com elevado tempo de análise e várias etapas de pré-concentração. Neste trabalho, procedimentos alternativos de extração e pré-concentração para analise multiresíduo de pesticida em água, frutas e tubérculos foram desenvolvidos. A eletroforese capilar em seu modo MEKC em condições de alto e baixo fluxo eletrosmótico foi empregado para a otimização da separação de diferentes classes de pesticidas (triazinas, organofosforado, carbendamidazóis, feniluréia e carbamatos). A composição do eletrólito de separação otimizada para condições de alto EOF foi: 10 mmol L-1 de tetraborato de sódio (pH 9,3),50 mmol L-1 de SDS e 5% etanol e 5% propanol, enquanto que para condições de baixo EOF foi: 10 mmol L-1 de tampão fosfato (pH 2,5), 25 mmol L-1 de SDS e 10% metanol. Estratégias de pré-concetração on-line conhecida como sweeping (SW) e stacking nos modos de migração reversa das micelas (SRMM) e migração reversa com um plug de água (SWR) bem como as suas versões modificadas foram avaliadas, obtendo fatores de pré-concetração de variaram de 2,6 a 19 para o SW, 2,9 a 15 para o SRW e de 5,5 a 15 para o método SRMM modificado. Varias metodologias de extração envolvendo extração em fase sólida (SPE) e extração líquido-líquido (LLE) foram testada. A estratégia de extração por cloud point foi aplicada a uma amostra de abacaxi. O procedimento denominado dispersão da matriz em fase sólida (sigla inglesa MSPD), que minimiza o uso de solventes orgânicos e é de fácil implementação foi aplicado a amostras de cenoura. A combinação do SPE off-line e das estratégias de pré-concentração anteriormente mencionadas permitiram a determinação de alguns pesticidas na concentração de 0,1 &#181;g L-1 em amostras de água potável. O método de extração e clean-up MSPD seguida da análise de MEKC em alto EOF foi otimizado e algumas figuras de mérito foram estabelecidas baseados em protocolos de validação para análise de pesticidas (IAEA-FAO). Boa linearidade (r > 0,99) foi obtido para todos os pesticidas estudados, exceto para linuron e dimetoato. A precisão do método foi estimada através de testes de recuperação. Dois níveis de fortificação foram utilizados para a avaliação, foram obtidos recuperações de 51 a 89 % para o nível mais baixo e 67 a 100% para o maior nível. Foi obtida uma boa precisão intraensaio (CV < 15%). O método otimizado foi aplicado para análise multiresíduo de cenouras. Uma amostra adquirida no comércio local foi quantificada encontrando-se 0,88 mg kg-1 de simazina, 0,13 mg kg-1 de atrazina e 0,08 mg kg-1 de propazina. / The use of pesticides constitutes an important aspect of modem agriculture, with unquestionable impact on crop production. However, food contamination by pesticide residues is a serious risk for the consumer. The determination of pesticide residues in food usually involves laborious procedures, with time consuming sample clean up and preconcentration steps prior to the analysis. In this work, alternative methodologies for extraction, pre-concentration and analysis of pesticide multi-residue in water, fruits and tubers were developed. Capillary electrophoresis in its micellar mode (micellar electrokinetic Chromatography, MEKC) under low and high electroosmotic flow (EOF) conditions was used for the separation of pesticides from different chemical classes (triazines, organophosphorous, carbendamidazols, phenilurea and carbamates). Optimized electrolyte compositions were: high EOF - 10 mmol L-1 tetraborate (pH 9.3), 50 mmol L-1 SDS, 5 % ethanol and 5 % propanol; low EOF - 10 mmol L-1 phosphate buffer (pH 2.5), 25 mmol L-1 SDS and 10 % methanol. On-line preconcentration strategies for MEKC known as sweeping (SW) and stacking with reverse migrating micelles with (SRW) and without (SRMM) a plug of water prior to the sample plug as well as modified versions of SRW and RMM were evaluated and contrasted in terms of signal enhancement factor (peak height ratios) Signal enhancement factors for SW varied from 2,6 to 19 for SRW from 2,9 to 15, whereas for modified-SRMM from 5,5 to 15. Among the extraction methodologies, several procedures involving solid-phase extraction (SPE) and liquid-liquid extraction (LLE) were tested. It is worth mentioning a strategy based upon cloud point extraction, which was applied to pineapple samples and a procedure denominated matrix solid-phase dispersion (MSPD), which combines low cost, saves in solvents and easy implementation, applied to carrots. The combination of off-line SPE and the above mentioned on-line preconcentration strategies allowed the determination of selected pesticides in the 0.1 &#181;/L level (drinking water sample). A complete methodology involving MSPD for extraction and sample clean-up followed by MEKC in high EOF was optimized and a few figures of merit were established based on method validation protocols for pesticide analysis (IAEA-FAO). Good linearity (r>0.99) was obtained for all pesticides under investigation, except for linuron and dimetoate. The method accuracy was estimated by recovery tests. Two level standard spiking were conducted with recoveries of 51 to 89 % for the lowest level and 67 to 100 % for the highest level. Acceptable intra-day precision was obtained (CV < 15 %). The optimized method was applied to the analysis of multi-residue pesticides in carrots. In a sample acquired in a local grocery store an unusual amount of triazines was found: simazine (O,88 mg/kg), atrazine (0,13 mg/kg) and propazine (0,088mg/kg).