The potential for monitoring and control of insect pests in Southern Hemisphere forestry plantations using semiochemicals

Southern Hemisphere plantation forestry has grown substantially over the past few decades and will play an increasing role in fibre production and carbon sequestration in future. The sustainability of these plantations is, however, increasingly under pressure from introduced pests. This pressure requires an urgent and matching increase in the speed and efficiency at which tools are developed to monitor and control these pests. To consider the potential role of semiochemicals to address the need for more efficient pest control in Southern Hemisphere plantations, particularly by drawing from research in other parts of the world. Semiochemical research in forestry has grown exponentially over the last 40 years but has been almost exclusively focussed on Northern Hemisphere forests. In these forests, semiochemicals have played an important role to enhance the efficiency of integrated pest management programmes. An analysis of semiochemical research from 1970 to 2010 showed a rapid increase over time. It also indicated that pheromones have been the most extensively studied type of semiochemical in forestry, contributing to 92% of the semiochemical literature over this period, compared with research on plant kairomones. This research has led to numerous applications in detection of new invasions, monitoring population levels and spread, in addition to controlling pests by mass trapping or disrupting of aggregation and mating signals. The value of semiochemicals as an environmentally benign and efficient approach to managing forest plantation pests in the Southern Hemisphere seems obvious. There is, however, a lack of research capacity and focus to optimally capture this opportunity. Given the pressure from increasing numbers of pests and reduced opportunities to use pesticides, there is some urgency to develop semiochemical research capacity.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Biological control of western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), in gerberas, chrysanthemums and roses

Frankliniella occidentalis (Pergande), western flower thrips (WFT), is a major worldwide pest of vegetables and ornamental crops. The biology of WFT was examined on gerberas, chrysanthemums and roses in relation to plant stage (flowering and non-flowering), pupation site, soil moisture and plant parts often inhabited by adult and immature thrips. Four foliage thrips predators ( Transeius montdorensis (Schicha), Orius armatus (Gross), Mallada signata (Schneider) and Neoseiulus cucumeris (Oudemans)) and three soil predators ( Geolaelaps aculeifer (Canestrini), Steinernema feltiae (Filipjev) and Dalotia coriaria (Kraatz)) were studied to determine their ability to reduce the numbers of WFT on gerberas, chrysanthemums and roses. There was no difference in the number of adults that emerged from growing media of high or low moisture content on any host plant. There were also no differences in the total numbers of WFT recaptured from flowering gerberas, chrysanthemums or roses. However, about seven times the number of thrips were collected from flowering chrysanthemums compared with non-flowering chrysanthemums, indicating that the flowering plants were more suitable hosts. Of all thrips recollected, the greatest percentage was immature (larval and pupal) thrips (70%, 71% and 43%) on the flowers for gerberas, chrysanthemums and roses, respectively. The mean percentage of thrips that emerged as adults from the soil was very low (5.31.2, 8.52.9, 20.59.1 and 28.25.6%) on gerberas, flowering and non-flowering chrysanthemums, and roses, respectively. Simultaneous release of foliage and soil predators did not reduce the number of thrips beyond that caused by foliage predators alone. Of the foliage predators, T. montdorensis, O. armatus and N. cucumeris performed best, significantly reducing the numbers of adult and immature thrips on flowers and foliage by 30-99%. Further research is required to determine the most cost-effective rates of release in cut flower crops.

Evaluation of chlorothalonil and paraffinic oil alone and in combinations with registered fungicides for the control of yellow Sigatoka (Mycosphaerella musicola) of bananas in Northern Queensland, Australia

The efficacy of chlorothalonil and paraffinic oil alone and in combinations with the registered fungicides propiconazole, tebuconazole, difenoconazole, epoxiconazole and pyrimethanil was evaluated in a field experiment over two cropping cycles in 2013 and 2014 in Northern Queensland, Australia, for control of yellow Sigatoka (caused by Mycosphaerella musicola) of banana. The predominantly applied by the banana industry treatment mancozeb with paraffinic oil was included for comparison. The results from the two cropping cycles suggested that all chemicals used with paraffinic oil were as effective or more effective than when applied with chlorothalonil, and chlorothalonil alone. Difenoconazole and epoxiconazole with paraffinic oil followed by propiconazole with paraffinic oil were the most effective treatments. Pyrimethanil and tebuconazole plus chlorothalonil were the least effective treatments. None of the chemical treatments was phytotoxic or reduced yield.

The economic impact of diseases and parasitic problems in freshwater fish production

Diseases and parasitic problems could constitute significant economic losses in fish production if not controlled, thus the need to continue monitoring its prevalence. Based on field studies on feral and intensively raised fish at the Kainji Lake Research Institute Nigeria, some diseases and parasitic problems have been identified. These include; helminthiasis; fungal disease; protozoa which include Myxosoma sp., Myxobolus spp., Henneguya sp., Trichodina sp., Ichthopthrius sp. bacterial mainly Aeromonas sp., Pseudomonas sp., mechanical injuries; death due to unknown causes and economic assessment of myxosporidian infection. Suggestion for disease control in fish production are recommended

Designing conformational control of human tissue transglutaminase

Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.

Traditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.

One such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.

To better understand the effects of TG2’s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation’s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2’s activities are believed to elicit pathological effects.

Robust Control of Evolutionary Dynamics

The application of principles from evolutionary biology has long been used to gain new insights into the progression and clinical control of both infectious diseases and neoplasms. This iterative evolutionary process consists of expansion, diversification and selection within an adaptive landscape - species are subject to random genetic or epigenetic alterations that result in variations; genetic information is inherited through asexual reproduction and strong selective pressures such as therapeutic intervention can lead to the adaptation and expansion of resistant variants. These principles lie at the center of modern evolutionary synthesis and constitute the primary reasons for the development of resistance and therapeutic failure, but also provide a framework that allows for more effective control.

A model system for studying the evolution of resistance and control of therapeutic failure is the treatment of chronic HIV-1 infection by broadly neutralizing antibody (bNAb) therapy. A relatively recent discovery is that a minority of HIV-infected individuals can produce broadly neutralizing antibodies, that is, antibodies that inhibit infection by many strains of HIV. Passive transfer of human antibodies for the prevention and treatment of HIV-1 infection is increasingly being considered as an alternative to a conventional vaccine. However, recent evolution studies have uncovered that antibody treatment can exert selective pressure on virus that results in the rapid evolution of resistance. In certain cases, complete resistance to an antibody is conferred with a single amino acid substitution on the viral envelope of HIV.

The challenges in uncovering resistance mechanisms and designing effective combination strategies to control evolutionary processes and prevent therapeutic failure apply more broadly. We are motivated by two questions: Can we predict the evolution to resistance by characterizing genetic alterations that contribute to modified phenotypic fitness? Given an evolutionary landscape and a set of candidate therapies, can we computationally synthesize treatment strategies that control evolution to resistance?

To address the first question, we propose a mathematical framework to reason about evolutionary dynamics of HIV from computationally derived Gibbs energy fitness landscapes -- expanding the theoretical concept of an evolutionary landscape originally conceived by Sewall Wright to a computable, quantifiable, multidimensional, structurally defined fitness surface upon which to study complex HIV evolutionary outcomes.

To design combination treatment strategies that control evolution to resistance, we propose a methodology that solves for optimal combinations and concentrations of candidate therapies, and allows for the ability to quantifiably explore tradeoffs in treatment design, such as limiting the number of candidate therapies in the combination, dosage constraints and robustness to error. Our algorithm is based on the application of recent results in optimal control to an HIV evolutionary dynamics model and is constructed from experimentally derived antibody resistant phenotypes and their single antibody pharmacodynamics. This method represents a first step towards integrating principled engineering techniques with an experimentally based mathematical model in the rational design of combination treatment strategies and offers predictive understanding of the effects of combination therapies of evolutionary dynamics and resistance of HIV. Preliminary in vitro studies suggest that the combination antibody therapies predicted by our algorithm can neutralize heterogeneous viral populations despite containing resistant mutations.

Renal failure and the neural control of the kidney

Renal failure (RF) is associated with an over activation of the sympathetic nervous system. The aim of this thesis was to investigate the hypothesis that as the kidney progresses into RF there is an inappropriate and sustained activation of renal afferent nerves which results in a dysregulation of basal RSNA and reflexly controlled RSNA by the high and low pressure baroreceptors. Baroreflex gain curves for both RSNA and HR were generated in control and RF rats. This study clearly showed a blunted high-pressure baroreflex in RF rats, an impairment which was almost completely corrected by bilateral renal denervation. The integrity of the low-pressure cardiopulmonary receptors to inhibit RSNA was investigated using acute saline volume. Again, a blunted reflex sympatho-inhibition of RSNA was observed, which was corrected by renal denervation. Finally a functional study to examine how the renal excretory response to volume expansion differed in RF was carried out. This study revealed an impairment of the low-pressure baroreflex control of the sympathetic outflow. The result of these studies suggest that cisplatin induced RF initiates a neural signal from within the kidney, which over rides the normal reflex regulation of RSNA by the high and low – pressure baroreceptors and that this impairment in function can be normalised by renal denervation. This raises further questions as to the mechanisms involved in the afferent over activation arising from the diseased kidneys.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1) ) and high (10(7) PFU mL(-1) ) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1) , respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

Biological control of the fish louse in a rainbow trout fishery

Novel egg-laying boards were found to be effective in the biological control of the freshwater fish louse Argulus foliaceus in a 12.9 ha rainbow trout Oncorhynchus mykiss fishery which had a high prevalence and intensity of infection of juvenile parasites in the early spring of 1999. Approximately 228 000d during an extensive 14 week period of egg laying which peaked in June 1999. In contrast, only 1566 clutches were harvested in 2000, when egg laying activity showed a bi-modal distribution, peaking in May and again in July and August. iaceus on rainbow trout in consecutive years was 2.9 : 1 and 2.1 : 1. Estimates of the size of the female A. foliaceus population based on egg-laying activity in 1999 exceeded that derived from measurements of prevalence and intensity of infection, whereas in 2000, this was more in balance. A minimum temperature of 10 degree C was identified for egg laying, which occurred continuously from May to October in a broadly synchronous manner.. Copyright 2002 The Fisheries Society of the British Isles

Immune engagement thresholds for CD8+ T CELL control of yherpesvirus-driven B CELL

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatológicas), Universidade de Lisboa, Faculdade de Medicina, 2014