Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.

Effect of Hydrologic Restoration on the Habitat of The Cape Sable Seaside Sparrow, Annual Report of 2002-2003

After developing field sampling protocols and making a series of consultations with investigators involved in research in CSSS habitat, we determined that vegetationhydrology interactions within this landscape are best sampled at a combination of scales. At the finer scale, we decided to sample at 100 m intervals along transects that cross the range of habitats present, and at the coarser scale, to conduct an extensive survey of vegetation at sites of known sparrow density dispersed throughout the range of the CSSS. We initiated sampling in the first week of January 2003 and continued it through the last week of May. During this period, we established 6 transects, one in each CSSS subpopulation, completed topographic survey along the Transects A, C, D, and F, and sampled herb and shrub stratum vegetation, soil depth and periphyton along Transects A, and at 179 census points. We also conducted topographic surveys and completed vegetation and soil depth sampling along two of five transects used by ENP researchers for monitoring long-term vegetation change in Taylor Slough. We analyzed the data by summarizing the compositional and structural measures and by using cluster analysis, ordination, weighted averaging regression, and weighted averaging calibration. The mean elevation of transects decreased from north to south, and Transect F had greater variation than other transects. We identified eight vegetation assemblages that can be grouped into two broad categories, ‘wet prairie’ and ‘marsh’. In the 2003 survey, wet prairies were most dominant in the northeastern sub-populations, and had shorter inferred-hydroperiod, higher species richness and shallower soils than marshes, which were common in Subpopulations A, D, and the southernmost regions of Sub-population B. Most of the sites at which birds were observed during 2001 or 2002 had an inferred-hydroperiod of 120-150 days, while no birds were observed at sites with an inferred-hydroperiod less than 120 days or more than 300 days. Management-induced water level changes in Taylor Slought during the 1980’s and 1990’s appeared to elicit parallel changes in vegetation. The results described in detail in the following pages serve as a basis for evaluating and modifying, if necessary, the sampling design and analytical techniques to be used in the next three years of the project.

The rodent fauna of Long Pine Key, Everglades National Park: a comparison of habitat types

Rodents are often involved at several stages of trophic dynamics. Consequently they often play crucial roles in the structure and function of many complex ecological systems. This study sought to address the lack of baseline data concerning rodents in tropical areas, and south Florida in particular. Live trapping took place in the four major habitat types of the Long Pine Key area of Everglades National Park over the course of one year. I compared population structures and abundance of murid rodents in the four habitat types, and tested multiple weather variables for their effectiveness as predictors of rodent abundance. I found the Long Pine Key area to be depauperate in terms of species diversity. Each of the four species of rodent encountered favored a particular habitat type. The density of the understory vegetation and the avoidance of avian predators in particular appear to be the most important factors in the distribution and abundance of rodents in the Long Pine Key area of Everglades National Park.

Drying Times: Integrating Citizen Science to Examine Survival of Florida Largemouth Bass in a Coastal Refuge Habitat

In aquatic systems refuge habitats increase resistance to drying events and are necessary for maintaining populations in disturbed environments. However, reduced water availability and altered flow regimes threaten the existence and function of these habitats. To test refuge function I conducted a capture-mark-recapture (CMR) study, integrating citizen science angler sampling into fisheries-independent methods. The objectives of this study were twofold: 1) To determine the contribution of citizen science anglers to improving CMR research, and 2.) to quantify apparent survival of Florida Largemouth Bass, Micropterus salmoides floridanus, in a coastal refuge habitat across multiple years of drying severity. The inclusion of angler sampling was determined to be an effective and feasible method for increasing capture probability. Apparent survival of Florida Bass varied among hydrologic periods with lowest survival when marshes functionally dried (< 10 cm). Overall mortality from drying events increased with the duration of marsh drying upstream.

Selected soil properties and bacterial abundance in the glacier forefields 'Black Valley Transect' and 'Glacier Transect' on Larsemann Hills, East Antarctica

Antarctic glacier forefields are extreme environments and pioneer sites for ecological succession. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats, and new terrain is becoming exposed to soil formation and microbial colonization. However, only little is known about the impact of environmental changes on microbial communities and how they develop in connection to shifting habitat characteristics. In this study, using a combination of molecular and geochemical analysis, we determine the structure and development of bacterial communities depending on soil parameters in two different glacier forefields on Larsemann Hills, East Antarctica. Our results demonstrate that deglaciation-dependent habitat formation, resulting in a gradient in soil moisture, pH and conductivity, leads to an orderly bacterial succession for some groups, for example Cyanobacteria, Bacteroidetes and Deltaproteobacteria in a transect representing 'classical' glacier forefields. A variable bacterial distribution and different composed communities were revealed according to soil heterogeneity in a slightly 'matured' glacier forefield transect, where Gemmatimonadetes, Flavobacteria, Gamma- and Deltaproteobacteria occur depending on water availability and soil depth. Actinobacteria are dominant in both sites with dominance connected to certain trace elements in the glacier forefields.

The Effects of Organic Matter Amendments and Migratory Waterfowl on Greenhouse Gas and Nutrient Dynamics in Managed Coastal Plain Wetlands

Wetland ecosystems provide many valuable ecosystem services, including carbon (C) storage and improvement of water quality. Yet, restored and managed wetlands are not frequently evaluated for their capacity to function in order to deliver on these values. Specific restoration or management practices designed to meet one set of criteria may yield unrecognized biogeochemical costs or co-benefits. The goal of this dissertation is to improve scientific understanding of how wetland restoration practices and waterfowl habitat management affect critical wetland biogeochemical processes related to greenhouse gas emissions and nutrient cycling. I met this goal through field and laboratory research experiments in which I tested for relationships between management factors and the biogeochemical responses of wetland soil, water, plants and trace gas emissions. Specifically, I quantified: (1) the effect of organic matter amendments on the carbon balance of a restored wetland; (2) the effectiveness of two static chamber designs in measuring methane (CH4) emissions from wetlands; (3) the impact of waterfowl herbivory on the oxygen-sensitive processes of methane emission and coupled nitrification-denitrification; and (4) nitrogen (N) exports caused by prescribed draw down of a waterfowl impoundment.

The potency of CH4 emissions from wetlands raises the concern that widespread restoration and/or creation of freshwater wetlands may present a radiative forcing hazard. Yet data on greenhouse gas emissions from restored wetlands are sparse and there has been little investigation into the greenhouse gas effects of amending wetland soils with organic matter, a recent practice used to improve function of mitigation wetlands in the Eastern United States. I measured trace gas emissions across an organic matter gradient at a restored wetland in the coastal plain of Virginia to test the hypothesis that added C substrate would increase the emission of CH4. I found soils heavily loaded with organic matter emitted significantly more carbon dioxide than those that have received little or no organic matter. CH4 emissions from the wetland were low compared to reference wetlands and contrary to my hypothesis, showed no relationship with the loading rate of added organic matter or total soil C. The addition of moderate amounts of organic matter (< 11.2 kg m-2) to the wetland did not greatly increase greenhouse gas emissions, while the addition of high amounts produced additional carbon dioxide, but not CH4.

I found that the static chambers I used for sampling CH4 in wetlands were highly sensitive to soil disturbance. Temporary compression around chambers during sampling inflated the initial chamber CH4 headspace concentration and/or lead to generation of nonlinear, unreliable flux estimates that had to be discarded. I tested an often-used rubber-gasket sealed static chamber against a water-filled-gutter seal chamber I designed that could be set up and sampled from a distance of 2 m with a remote rod sampling system to reduce soil disturbance. Compared to the conventional design, the remotely-sampled static chambers reduced the chance of detecting inflated initial CH4 concentrations from 66 to 6%, and nearly doubled the proportion of robust linear regressions from 45 to 86%. The new system I developed allows for more accurate and reliable CH4 sampling without costly boardwalk construction.

I explored the relationship between CH4 emissions and aquatic herbivores, which are recognized for imposing top-down control on the structure of wetland ecosystems. The biogeochemical consequences of herbivore-driven disruption of plant growth, and in turn, mediated oxygen transport into wetland sediments, were not previously known. Two growing seasons of herbivore exclusion experiments in a major waterfowl overwintering wetland in the Southeastern U.S. demonstrate that waterfowl herbivory had a strong impact on the oxygen-sensitive processes of CH4 emission and nitrification. Denudation by herbivorous birds increased cumulative CH4 flux by 233% (a mean of 63 g CH4 m-2 y-1) and inhibited coupled nitrification-denitrification, as indicated by nitrate availability and emissions of nitrous oxide. The recognition that large populations of aquatic herbivores may influence the capacity for wetlands to emit greenhouse gases and cycle nitrogen is particularly salient in the context of climate change and nutrient pollution mitigation goals. For example, our results suggest that annual emissions of 23 Gg of CH4 y-1 from ~55,000 ha of publicly owned waterfowl impoundments in the Southeastern U.S. could be tripled by overgrazing.

Hydrologically controlled moist-soil impoundment wetlands provide critical habitat for high densities of migratory bird populations, thus their potential to export nitrogen (N) to downstream waters may contribute to the eutrophication of aquatic ecosystems. To investigate the relative importance of N export from these built and managed habitats, I conducted a field study at an impoundment wetland that drains into hypereutrophic Lake Mattamuskeet. I found that prescribed hydrologic drawdowns of the impoundment exported roughly the same amount of N (14 to 22 kg ha-1) as adjacent fertilized agricultural fields (16 to 31 kg ha-1), and contributed approximately one-fifth of total N load (~45 Mg N y-1) to Lake Mattamuskeet. Ironically, the prescribed drawdown regime, designed to maximize waterfowl production in impoundments, may be exacerbating the degradation of habitat quality in the downstream lake. Few studies of wetland N dynamics have targeted impoundments managed to provide wildlife habitat, but a similar phenomenon may occur in some of the 36,000 ha of similarly-managed moist-soil impoundments on National Wildlife Refuges in the southeastern U.S. I suggest early drawdown as a potential method to mitigate impoundment N pollution and estimate it could reduce N export from our study impoundment by more than 70%.

In this dissertation research I found direct relationships between wetland restoration and impoundment management practices, and biogeochemical responses of greenhouse gas emission and nutrient cycling. Elevated soil C at a restored wetland increased CO2 losses even ten years after the organic matter was originally added and intensive herbivory impact on emergent aquatic vegetation resulted in a ~230% increase in CH4 emissions and impaired N cycling and removal. These findings have important implications for the basic understanding of the biogeochemical functioning of wetlands and practical importance for wetland restoration and impoundment management in the face of pressure to mitigate the environmental challenges of global warming and aquatic eutrophication.

RELIBILITY ANALYSIS OF SIMPLE SLOPES AND SOIL-STRUCTURES WITH LINEAR LIMIT STATES

The first objective of this research was to develop closed-form and numerical probabilistic methods of analysis that can be applied to otherwise conventional methods of unreinforced and geosynthetic reinforced slopes and walls. These probabilistic methods explicitly include random variability of soil and reinforcement, spatial variability of the soil, and cross-correlation between soil input parameters on probability of failure. The quantitative impact of simultaneously considering the influence of random and/or spatial variability in soil properties in combination with cross-correlation in soil properties is investigated for the first time in the research literature. Depending on the magnitude of these statistical descriptors, margins of safety based on conventional notions of safety may be very different from margins of safety expressed in terms of probability of failure (or reliability index). The thesis work also shows that intuitive notions of margin of safety using conventional factor of safety and probability of failure can be brought into alignment when cross-correlation between soil properties is considered in a rigorous manner. The second objective of this thesis work was to develop a general closed-form solution to compute the true probability of failure (or reliability index) of a simple linear limit state function with one load term and one resistance term expressed first in general probabilistic terms and then migrated to a LRFD format for the purpose of LRFD calibration. The formulation considers contributions to probability of failure due to model type, uncertainty in bias values, bias dependencies, uncertainty in estimates of nominal values for correlated and uncorrelated load and resistance terms, and average margin of safety expressed as the operational factor of safety (OFS). Bias is defined as the ratio of measured to predicted value. Parametric analyses were carried out to show that ignoring possible correlations between random variables can lead to conservative (safe) values of resistance factor in some cases and in other cases to non-conservative (unsafe) values. Example LRFD calibrations were carried out using different load and resistance models for the pullout internal stability limit state of steel strip and geosynthetic reinforced soil walls together with matching bias data reported in the literature.

Nonlinear soil-structure interaction for buried pressure pipes under differential ground motion

Pipelines extend thousands of kilometers across wide geographic areas as a network to provide essential services for modern life. It is inevitable that pipelines must pass through unfavorable ground conditions, which are susceptible to natural disasters. This thesis investigates the behaviour of buried pressure pipelines experiencing ground distortions induced by normal faulting. A recent large database of physical modelling observations on buried pipes of different stiffness relative to the surrounding soil subjected to normal faults provided a unique opportunity to calibrate numerical tools. Three-dimensional finite element models were developed to enable the complex soil-structure interaction phenomena to be further understood, especially on the subjects of gap formation beneath the pipe and the trench effect associated with the interaction between backfill and native soils. Benchmarked numerical tools were then used to perform parametric analysis regarding project geometry, backfill material, relative pipe-soil stiffness and pipe diameter. Seismic loading produces a soil displacement profile that can be expressed by isoil, the distance between the peak curvature and the point of contraflexure. A simplified design framework based on this length scale (i.e., the Kappa method) was developed, which features estimates of longitudinal bending moments of buried pipes using a characteristic length, ipipe, the distance from peak to zero curvature. Recent studies indicated that empirical soil springs that were calibrated against rigid pipes are not suitable for analyzing flexible pipes, since they lead to excessive conservatism (for design). A large-scale split-box normal fault simulator was therefore assembled to produce experimental data for flexible PVC pipe responses to a normal fault. Digital image correlation (DIC) was employed to analyze the soil displacement field, and both optical fibres and conventional strain gauges were used to measure pipe strains. A refinement to the Kappa method was introduced to enable the calculation of axial strains as a function of pipe elongation induced by flexure and an approximation of the longitudinal ground deformations. A closed-form Winkler solution of flexural response was also derived to account for the distributed normal fault pattern. Finally, these two analytical solutions were evaluated against the pipe responses observed in the large-scale laboratory tests.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database

Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The ‘Biomes of Australian Soil Environments’ (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function.

Biodiversité des pollinisateurs dans les cannebergières : effets des pratiques culturales, du type de sol et de l'habitat naturel

Les pollinisateurs indigènes sont importants pour la production de canneberges. Ils sont pourtant menacés par les pratiques agricoles qui s’intensifient. Pour établir un plan de conservation efficace, une meilleure connaissance des facteurs intrinsèques aux exploitations est essentielle. L’objectif principal de cette étude était de déterminer la diversité et abondance des abeilles et syrphes retrouvées en cannebergières. Quinze fermes ont été échantillonnées selon la régie de culture, le type de sol et le type d’habitat naturel retrouvé près des cannebergières. Des pièges-bols disposés en transect ainsi que l’utilisation de filet entomologique ont été utilisés pour échantillonner les pollinisateurs. Au total, 135 espèces de pollinisateurs indigènes ont été capturées en 2013 et 2014. Le type de sol ainsi que l’habitat naturel influençaient les communautés de pollinisateurs, alors que la régie de culture ne les affectait pas. L’abeille Melitta americana, spécialiste du pollen de canneberge, a été capturée plus souvent dans les tourbières.

Wheats developed for high yield on stored soil moisture have deep vigorous root systems

Many rainfed wheat production systems are reliant on stored soil water for some or all of their water inputs. Selection and breeding for root traits could result in a yield benefit; however, breeding for root traits has traditionally been avoided due to the difficulty of phenotyping mature root systems, limited understanding of root system development and function, and the strong influence of environmental conditions on the phenotype of the mature root system. This paper outlines an international field selection program for beneficial root traits at maturity using soil coring in India and Australia. In the rainfed areas of India, wheat is sown at the end of the monsoon into hot soils with a quickly receding soil water profile; in season water inputs are minimal. We hypothesised that wheat selected and bred for high yield under these conditions would have deep, vigorous root systems, allowing them to access and utilise the stored soil water at depth around anthesis and grain-filling when surface layers were dry. The Indian trials resulted in 49 lines being sent to Australia for phenotyping. These lines were ranked against 41 high yielding Australian lines. Variation was observed for deep root traits e.g. in eastern Australia in 2012, maximum depth ranged from 118.8 to 146.3 cm. There was significant variation for root traits between sites and years, however, several Indian genotypes were identified that consistently ranked highly across sites and years for deep rooting traits.

Assessment of iodine intake and related cognitive function impairments in elementary schoolchildren from Terceira Island, Azores (Portugal)

Dissertação de Mestrado, Ciências Biomédicas, 28 de Junho de 2016, Universidade dos Açores.

Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation

Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.

Burrows with Chimneys of the Fiddler Crab Uca thayeri: Construction, Occurrence, and Function

Joao B. L. Gusmao-Junior, Glauco B. O. Machado, and Tania M. Costa (2012) Burrows with chimneys of the fiddler crab Uca thayeri: construction, occurrence, and function. Zoological Studies 51(5): 598-605. Building of soil structures is observed in a variety of semi-terrestrial crustaceans. In fiddler crabs (Genus Uca), this behavior occurs in several species, some of which build structures that are largely ornamental and others construct barriers that are apparently for defense. Although there is a relative abundance of studies on this type of behavior in Uca, the relationship between the social context and the occurrence of these structures remains poorly studied. Thus, this study attempted to analyze in detail the construction, occurrence, and function of mud chimneys built by the fiddler crab Uca thayeri; these sedimentary structures are possibly associated with burrow defense. Field investigations and laboratory experiments were conducted. Both sexes were often found in burrows with chimneys; however, laboratory experiments showed that only females actively built and maintained chimneys, with some difference in the morphology of these structures between sexes. The social context had little influence on the construction of chimneys, which showed that the stimulus for constructing chimneys could be endogenous. Our results suggest that burrows with chimney of U. thayeri may have functions other than defense, and may act in regulating the internal conditions of the burrow, as observed in other crustaceans with such building behavior. http://zoolstud.sinica.edu.tw/Journals/51.5/598.pdf

Térmitas como bioindicadores de qualidade de habitat na caatinga, Brasil: há uma sintonia entre as variáveis estruturais dos habitats e as taxocenoses amostradas?

The composition of termite assemblages was analyzed at three Caatinga sites of the Seridó Ecological Station, located in the municipality of Serra Negra do Norte, in the state of Rio Grande do Norte, Brazil. These sites have been subjected to selective logging, and cleared for pasture and farming. A standardized sampling protocol for termite assemblages (30h/person/site) was conducted between September 2007 and February 2009. At each site we measured environmental variables, such as soil granulometry, pH and organic matter, necromass stock, vegetation height, tree density, stem diameter at ankle height (DAH) and the largest and the smallest crown width. Ten species of termites, belonging to eight genera and three families, were found at the three experimental sites. Four feeding-groups were sampled: wood-feeders, soil-feeders, wood-soil interface feeders and leaf-feeders. The wood-feeders were dominant in number of species and number of encounters at all sites. In general, the sites were not significantly different in relation to the environmental variables measured. The same pattern was observed for termite assemblages, where no significant differences in species richness, relative abundance and taxonomic and functional composition were observed between the three sites. The agreement between the composition of assemblages and environmental variables reinforces the potential of termites as biological indicators of habitat quality