Development of Locust Bean Gum Nanoparticles for protein delivery

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Inhalable antitubercular therapy mediated by locust bean gum microparticles

Tuberculosis remains a major global health problem and alternative therapeutic approaches are needed. Considering the high prevalence of lung tuberculosis (80% of cases), the pulmonary delivery of antitubercular drugs in a carrier system capable of reaching the alveoli, being recognised and phagocytosed by alveolar macrophages (mycobacterium hosts), would be a significant improvement to current oral drug regimens. Locust bean gum (LBG) is a polysaccharide composed of galactose and mannose residues, which may favour specific recognition by macrophages and potentiate phagocytosis. LBG microparticles produced by spray-drying are reported herein for the first time, incorporating either isoniazid or rifabutin, first-line antitubercular drugs (association efficiencies >82%). Microparticles have adequate theoretical properties for deep lung delivery (aerodynamic diameters between 1.15 and 1.67 μm). The cytotoxic evaluation in lung epithelial cells (A549 cells) and macrophages (THP-1 cells) revealed a toxic effect from rifabutin-loaded microparticles at the highest concentrations, but we may consider that these were very high comparing with in vivo conditions. LBG microparticles further evidenced strong ability to be captured by macrophages (percentage of phagocytosis >94%). Overall, the obtained data indicated the potential of the proposed system for tuberculosis therapy.

Resistance of Bt transgenic maize to lesser cornstalk borer (Lepidoptera: Pyralidae).

To determine if Bt maize seedlings are protected against lesser cornstalk borer damage, Bt hybrids at the 3 and 4 leaf stages were tested under temperatures between 20-32oC and artificial infestation. A high level of resistance was reported in all Bt maize as expressed by larval survival, larval weight, damage score, and number of surviving plants. All Bt maize protected plants against lesser cornstalk borer damage. Also, the resistance present in the Bt maize was not affected by daytime temperature.

EVALUATION AND MONITORING OF ASH (FRAXINUS SPP.) TOLERANT TO LONG-TERM EMERALD ASH BORER (AGRILUS PLANIPENNIS [COLEOPTERA: BUPRESTIDAE]) EXPOSURE

Invasive insects that successfully establish in introduced areas can significantly alter natural communities. These pests require specific establishment criteria (e.g. host suitability) that, when known, can help quantify potential damage to infested areas. Emerald ash borer (Agrilus planipennis [Coleoptera: Buprestidae]) is an invasive phloem-feeding pest which is responsible for the death of millions of ash trees (Fraxinus spp. L.). Over 200 surviving ash trees were previously identified in the Huron-Clinton Metroparks located in southeast Michigan. Trees were assessed over a four year period and a hierarchical cluster analysis was performed on dieback, vigor, and presence of signs and symptoms, in order to place trees into one of three tolerance groups. The clustering of trees with different responses to emerald ash borer attack suggests that there are different tolerance levels in North American ash trees in southeastern Michigan, and these groups were designated as apparently tolerant, not tolerant and intermediate tolerance. Adult landing rates and evidence of adult emergence were significantly lower in the apparently tolerant group compared with the not tolerant group, but larval survival from eggs placed on trees did not differ between tolerance groups. Therefore, it appears that apparently tolerant trees survive because they are less attractive to adult beetles which results in fewer eggs being laid on them. Trees in the apparently tolerant group remained of higher vigor over the four years of the study. North American ash may survive the emerald ash borer epidemic due to natural variation and inherent resistance regardless of the lack of co-evolutionary history with emerald ash borer.

Cold acclimation of chill coma mechanisms in the locust CNS

Temperature has profound effects on the neural function and behaviour of insects. When exposed to low temperature, migratory locusts (Locusta migratoria) enter chill coma (neuromuscular paralysis) and can resume normal body functions after returning to normal temperature. Our laboratory has studied phenomena underlying environmental stress-induced comas in locusts and found that they are associated with a sudden loss of K+ homeostasis and also a temporary electrical silence in the central nervous system (CNS). However, the mechanisms underlying chill coma entry and recovery are not well understood, particularly the role of the CNS has not been determined. Here, I investigated neural function during chill coma in the locust by measuring electrical activity in the CNS. As pre-exposure to moderately low temperatures, either chronically (cold acclimation) or acutely (rapid cold hardening; RCH), has been found to improve the insect’s cold tolerance, I also determined cold acclimation and RCH protocols that will improve the locust's cold tolerance and whether these protocols affect neural shutdown during chill coma in the locust. With an implanted thermocouple in the thorax, I determined the temperature associated with a loss of responsiveness (CTmin) in intact male adult locusts. In parallel experiments, I recorded field potential (FP) in the metathoracic ganglion (MTG) in semi-intact preparations to determine the temperature that would induce neural shutdown. I found that acclimation at 10 ˚C and RCH at 4 ˚C reduced chill coma recovery time (CCRT) in intact animal preparations and RCH at 4 ˚C for 4 hours reduced the temperature at neural shutdown in semi-intact preparations. These results suggest that pre-exposure to cold can improve the locust's resistance to chill coma and support the notion that the CNS has a role in determining entry into and exit from chill coma in locusts.

Bollywood Extras

"Bollywood Extras" has been described as a bit like Vladimir Nabokov's "Lolita" (1955) meets Nathanael West's "Day of the Locust" (1939). Unlike those two classics, this new novel by Dr D. Bruno Starrs is set in 21st Century India's Hindi-language film industry epicenter known as 'Bollywood', Mumbai's answer to America's 'Hollywood'. And there is another major difference: the story (i.e. the narrative interaction between an American Extras Casting Agent, 'Dr Arden Pyle', an under-age wannabe Bollywood starlet, 'Chandy', and the despicable but wealthy Indian man who stalks her, 'Ishmail'), is all staged against a backdrop of rabid religious terrorism. Written with the unique black comedic and literary flair Dr D. Bruno Starrs is renown for, this, his 3rd full-length novel, boldly captures the feel of Mumbai and the small-time players in its big-time film industry, with style, humor and originality. Tom Flood, winner of the Miles Franklin Literary Award and founder of Flood Manuscripts, said this of the novel's second last draft: "What is best about 'Bollywood Extras' will likely be its albatross in the sliced bread world of mainstream publishing. Three strengths that make the work what it is - the length, the style, the intellectual capital - will be three strikes against it when it comes to the money. While I delight in rich language and agile invention, I've given you the reasons the trade ('legit') presses won't take 'Bollywood Extras'. Do I think you should you change it? No. I like it. Why ruin an interesting work for money?" As a professional assessor, Flood did, of course, offer many suggestions and these were duly implemented, although the author steadfastly adhered to the style he had already cemented, thus not changing in anyway what Flood refers to as its three strengths. Dr Starrs knew (having accessed Flood's professional services previously) that a 'Thumbs Up' from the best manuscript assessor in the country meant that Bollywood Extras was ready to be birthed. So, here it is: Dr Starrs has delivered his 3rd baby and 'christened' it "Bollywood Extras"!

Response to comments on "Productivity is a poor predictor of plant species richness"

Pan et al. claim that our results actually support a strong linear positive relationship between productivity and richness, whereas Fridley et al. contend that the data support a strong humped relationship. These responses illustrate how preoccupation with bivariate patterns distracts from a deeper understanding of the multivariate mechanisms that control these important ecosystem properties.

Abundance of introduced species at home predicts abundance away in herbaceous communities

Many ecosystems worldwide are dominated by introduced plant species, leading to loss of biodiversity and ecosystem function. A common but rarely tested assumption is that these plants are more abundant in introduced vs. native communities, because ecological or evolutionary-based shifts in populations underlie invasion success. Here, data for 26 herbaceous species at 39 sites, within eight countries, revealed that species abundances were similar at native (home) and introduced (away) sites – grass species were generally abundant home and away, while forbs were low in abundance, but more abundant at home. Sites with six or more of these species had similar community abundance hierarchies, suggesting that suites of introduced species are assembling similarly on different continents. Overall, we found that substantial changes to populations are not necessarily a pre-condition for invasion success and that increases in species abundance are unusual. Instead, abundance at home predicts abundance away, a potentially useful additional criterion for biosecurity programmes.

The influence of temperature and introduction point on the detection of Rhyzopertha dominica in stored grain

The presence of insect pests in grain storages throughout the supply chain is a significant problem for farmers, grain handlers, and distributors world-wide. Insect monitoring and sampling programmes are used in the stored grains industry for the detection and estimation of pest populations. At the low pest densities dictated by economic and commercial requirements, the accuracy of both detection and abundance estimates can be influenced by variations in the spatial structure of pest populations over short distances. Geostatistical analysis of Rhyzopertha dominica populations in 2 and 3 dimensions showed that insect numbers were positively correlated over short (0.5 cm) distances, and negatively correlated over longer (.10 cm) distances. At 35 C, insects were located significantly further from the grain surface than at 25 and 30 C. Dispersion metrics showed statistically significant aggregation in all cases. The observed heterogeneous spatial distribution of R. dominica may also be influenced by factors such as the site of initial infestation and disturbance during handling. To account for these additional factors, I significantly extended a simulation model that incorporates both pest growth and movement through a typical stored-grain supply chain. By incorporating the effects of abundance, initial infestation site, grain handling, and treatment on pest spatial distribution, I developed a supply chain model incorporating estimates of pest spatial distribution. This was used to examine several scenarios representative of grain movement through a supply chain, and determine the influence of infestation location and grain disturbance on the sampling intensity required to detect pest infestations at various infestation rates. This study has investigated the effects of temperature, infestation point, and grain handling on the spatial distribution and detection of R. dominica. The proportion of grain infested was found to be dependent upon abundance, initial pest location, and grain handling. Simulation modelling indicated that accounting for these factors when developing sampling strategies for stored grain has the potential to significantly reduce sampling costs while simultaneously improving detection rate, resulting in reduced storage and pest management cost while improving grain quality.

Life-history constraints in grassland plant species : a growth-defence trade-off is the norm

Plant growth can be limited by resource acquisition and defence against consumers, leading to contrasting trade-off possibilities. The competition-defence hypothesis posits a trade-off between competitive ability and defence against enemies (e.g. herbivores and pathogens). The growth-defence hypothesis suggests that strong competitors for nutrients are also defended against enemies, at a cost to growth rate. We tested these hypotheses using observations of 706 plant populations of over 500 species before and following identical fertilisation and fencing treatments at 39 grassland sites worldwide. Strong positive covariance in species responses to both treatments provided support for a growth-defence trade-off: populations that increased with the removal of nutrient limitation (poor competitors) also increased following removal of consumers. This result held globally across 4 years within plant life-history groups and within the majority of individual sites. Thus, a growth-defence trade-off appears to be the norm, and mechanisms maintaining grassland biodiversity may operate within this constraint.

Regional contingencies in the relationship between aboveground biomass and litter in the world’s grasslands

Based on regional-scale studies, aboveground production and litter decomposition are thought to positively covary, because they are driven by shared biotic and climatic factors. Until now we have been unable to test whether production and decomposition are generally coupled across climatically dissimilar regions, because we lacked replicated data collected within a single vegetation type across multiple regions, obfuscating the drivers and generality of the association between production and decomposition. Furthermore, our understanding of the relationships between production and decomposition rests heavily on separate meta-analyses of each response, because no studies have simultaneously measured production and the accumulation or decomposition of litter using consistent methods at globally relevant scales. Here, we use a multi-country grassland dataset collected using a standardized protocol to show that live plant biomass (an estimate of aboveground net primary production) and litter disappearance (represented by mass loss of aboveground litter) do not strongly covary. Live biomass and litter disappearance varied at different spatial scales. There was substantial variation in live biomass among continents, sites and plots whereas among continent differences accounted for most of the variation in litter disappearance rates. Although there were strong associations among aboveground biomass, litter disappearance and climatic factors in some regions (e.g. U.S. Great Plains), these relationships were inconsistent within and among the regions represented by this study. These results highlight the importance of replication among regions and continents when characterizing the correlations between ecosystem processes and interpreting their global-scale implications for carbon flux. We must exercise caution in parameterizing litter decomposition and aboveground production in future regional and global carbon models as their relationship is complex.

Predicting invasion in grassland ecosystems: is exotic dominance the real embarrassment of richness?

Invasions have increased the size of regional species pools, but are typically assumed to reduce native diversity. However, global-scale tests of this assumption have been elusive because of the focus on exotic species richness, rather than relative abundance. This is problematic because low invader richness can indicate invasion resistance by the native community or, alternatively, dominance by a single exotic species. Here, we used a globally replicated study to quantify relationships between exotic richness and abundance in grass-dominated ecosystems in 13 countries on six continents, ranging from salt marshes to alpine tundra. We tested effects of human land use, native community diversity, herbivore pressure, and nutrient limitation on exotic plant dominance. Despite its widespread use, exotic richness was a poor proxy for exotic dominance at low exotic richness, because sites that contained few exotic species ranged from relatively pristine (low exotic richness and cover) to almost completely exotic-dominated ones (low exotic richness but high exotic cover). Both exotic cover and richness were predicted by native plant diversity (native grass richness) and land use (distance to cultivation). Although climate was important for predicting both exotic cover and richness, climatic factors predicting cover (precipitation variability) differed from those predicting richness (maximum temperature and mean temperature in the wettest quarter). Herbivory and nutrient limitation did not predict exotic richness or cover. Exotic dominance was greatest in areas with low native grass richness at the site- or regional-scale. Although this could reflect native grass displacement, a lack of biotic resistance is a more likely explanation, given that grasses comprise the most aggressive invaders. These findings underscore the need to move beyond richness as a surrogate for the extent of invasion, because this metric confounds monodominance with invasion resistance. Monitoring species' relative abundance will more rapidly advance our understanding of invasions.

Anthropogenic-based regional-scale factors most consistently explain plot-level exotic diversity in grasslands

Aim Evidence linking the accumulation of exotic species to the suppression of native diversity is equivocal, often relying on data from studies that have used different methods. Plot-level studies often attribute inverse relationships between native and exotic diversity to competition, but regional abiotic filters, including anthropogenic influences, can produce similar patterns.We seek to test these alternatives using identical scale-dependent sampling protocols in multiple grasslands on two continents. Location Thirty-two grassland sites in North America and Australia. Methods We use multiscale observational data, collected identically in grain and extent at each site, to test the association of local and regional factors with the plot-level richness and abundance of native and exotic plants. Sites captured environmental and anthropogenic gradients including land-use intensity, human population density, light and soil resources, climate and elevation. Site selection occurred independently of exotic diversity, meaning that the numbers of exotic species varied randomly thereby reducing potential biases if only highly invaded sites were chosen. Results Regional factors associated directly or indirectly with human activity had the strongest associations with plot-level diversity. These regional drivers had divergent effects: urban-based economic activity was associated with high exotic : native diversity ratios; climate- and landscape-based indicators of lower human population density were associated with low exotic : native ratios. Negative correlations between plot-level native and exotic diversity, a potential signature of competitive interactions, were not prevalent; this result did not change along gradients of productivity or heterogeneity. Main conclusion We show that plot-level diversity of native and exotic plants are more consistently associatedwith regional-scale factors relating to urbanization and climate suitability than measures indicative of competition. These findings clarify the long-standing difficulty in resolving drivers of exotic diversity using single-factor mechanisms, suggesting that multiple interacting anthropogenic-based processes best explain the accumulation of exotic diversity in modern landscapes.

Eutrophication weakens stabilizing effects of diversity in natural grasslands

Studies of experimental grassland communities have demonstrated that plant diversity can stabilize productivity through species asynchrony, in which decreases in the biomass of some species are compensated for by increases in others. However, it remains unknown whether these findings are relevant to natural ecosystems, especially those for which species diversity is threatened by anthropogenic global change. Here we analyse diversity-stability relationships from 41 grasslands on five continents and examine how these relationships are affected by chronic fertilization, one of the strongest drivers of species loss globally. Unmanipulated communities with more species had greater species asynchrony, resulting in more stable biomass production, generalizing a result from biodiversity experiments to real-world grasslands. However, fertilization weakened the positive effect of diversity on stability. Contrary to expectations, this was not due to species loss after eutrophication but rather to an increase in the temporal variation of productivity in combination with a decrease in species asynchrony in diverse communities. Our results demonstrate separate and synergistic effects of diversity and eutrophication on stability, emphasizing the need to understand how drivers of global change interactively affect the reliable provisioning of ecosystem services in real-world systems.

Herbivores and nutrients control grassland plant diversity via light limitation

Human alterations to nutrient cycles1, 2 and herbivore communities3, 4, 5, 6, 7 are affecting global biodiversity dramatically2. Ecological theory predicts these changes should be strongly counteractive: nutrient addition drives plant species loss through intensified competition for light, whereas herbivores prevent competitive exclusion by increasing ground-level light, particularly in productive systems8, 9. Here we use experimental data spanning a globally relevant range of conditions to test the hypothesis that herbaceous plant species losses caused by eutrophication may be offset by increased light availability due to herbivory. This experiment, replicated in 40 grasslands on 6 continents, demonstrates that nutrients and herbivores can serve as counteracting forces to control local plant diversity through light limitation, independent of site productivity, soil nitrogen, herbivore type and climate. Nutrient addition consistently reduced local diversity through light limitation, and herbivory rescued diversity at sites where it alleviated light limitation. Thus, species loss from anthropogenic eutrophication can be ameliorated in grasslands where herbivory increases ground-level light.