Using High Throughput Genomic Sequencing to Predict Ecological Impacts on Sea Turtle Populations

Marine turtles are long-lived, migratory vertebrates that encounter a variety of human and natural stressors throughout their lives. Understanding the biology and threats of these animals is challenging because they are hard to observe, and can migrate across whole ocean basins. Minimally invasive sampling techniques (e.g., blood samples) allow us to learn about their physiology, genetics, and the environmental conditions they have experienced. In this project, we developed a novel method to extract the RNA from whole green and loggerhead turtle blood from animals inhabiting a variety of sites across the Pacific Ocean. Some habitats are more pristine, while others have been heavily altered by humans. These samples will be used for high throughput genomic sequencing to provide data on the gene expression, genetic relatedness, and functional genomics of these animals. First, we will look at the genes that are up-regulated in animals inhabiting polluted environments to understand contaminant impacts on marine turtle health. Secondly, we will identify single nucleotide polymorphisms that will be used as tools in future studies to examine fine-scale structure and adaptive variation in future studies. We then pair these data with other data from our research group on sex, size, migration, and foraging ecology. These approaches allow us to generate large amounts of data that can be used for a variety of scientific and conservation studies from small samples. This allows us to sample the animals while having little to no impact, and to monitor health and physiology in live populations over time. This will provide a better understanding of threats to sea turtle populations that can be used by both conservation biologists and ecologists in protecting habitats of sea turtles worldwide

RNA Sequencing (RNA-Seq) Reveals Extremely Low Levels of Reticulocyte-Derived Globin Gene Transcripts in Peripheral Blood From Horses (Equus caballus) and Cattle (Bos taurus)

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Conclusion: By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species

Immunophenotypes of Australian Sea Lion Pups (Neophoca cinerea) Developing in the Context of Hookworm Infection (Uncinaria sanguinis)

Pinnipeds are exposed to anthropogenic and environmental stressors, including disease, and these can, in turn, affect their immune system, health, and viability. The endemic Australian sea lion (Neophoca cinerea) population was impacted severely by commercial sealing in the 18th and 19th centuries and is now threatened by high pup mortality, such that hookworm disease (Uncinaria sanguinis) in pups has been identified as likely contributor to a declining population. To understand how the immune system functions, protects wild hosts from disease or causes immunopathology, it is crucial to bring our immunological toolbox into wild populations to measure immune phenotypes in the context of disease and other potential sources of heterogeneity, such as age. The immune profiles of Australian sea lion pups were assessed using serial blood samples collected from N. cinerea pups from two colonies located in South Australia, i.e. Seal Bay (n = 38) and Dangerous Reef (n = 34). One cohort of pups from Dangerous Reef was treated with ivermectin to provide a hookworm-negative cohort, allowing evaluation of the impact of this endemic infection and significant disease on immunophenotype. We measured indicators of health involved in innate and Th1/Th2 responses (IL-6, TNFα, IFNγ, IL-4, IL-10, lysozyme, acute-phase proteins and IgG) controlling for demographic factors such as development and colony or hookworm intensity season. Preliminary attempts to measure IL-4 and IFNγ in Australian sea lion pups blood samples revealed very low copy numbers using qPCR and digital droplet PCR methods, suggesting further investigations of these profiles. We found immunophenotypic differences associated with age, colony and disease status, highlighting some potentially promising indicators of health in the Australian sea lion

Intercellular production of Hydrogen peroxide in Solanum lycopersicum: an immune response to Salmonella infection

Produce associated gastroenteritis in recent years has become more associated with fruits and vegetables rather than raw meat and dairy, with thousands of deaths per year recorded. Outbreaks of Salmonella have been reported from a multitude of vegetables such as lettuce, spinach and tomatoes. Many of these outbreaks are the result of infection via non-typhoid Salmonella species such as Salmonella enterica sv. Typhimurium. The ecology of Salmonella as a human pathogen via vegetable hosts is increasingly being studied, however much is still unknown. Studies have shown that Salmonella may proliferate in both the rhizosphere and phyllosphere of the tomato plant Solanum lycopersiscum. In tomatoes, Salmonella has varying levels of proliferation success depending on the variety of tomato in question. Previous studies had shown that tomatoes of the cherry variety were significantly more resistant to Salmonella proliferation than larger varieties. S. lycopersicum var. cerasiforme and var. alicante were inoculated with Salmonella enterica (LT2) sv. Typhimurium and allowed to incubate for 72 hours. Following this, tomatoes were homogenised and samples plated on XLD. This study found that Salmonella ability to proliferate in var. cerasiforme was significantly different (α=0.05) when compared to var. alicante. With Salmonella proliferating up to a 1 log more in var. alicante than in var. cerasiforme. This study suggested that this difference was due to an innate difference in immune response efficacy between var. cerasiforme and var. alicante. Studies regarding plant immune responses have shown that production of hydrogen peroxide is a common response to microbial attack. Using a novel methodology, this study extracted the intercellular fluid from the tomato fruit in both var. alicante and var. cerasiforme. Analysis via fluorometric quantification found that var. cerasiforme contained significantly higher amounts of hydrogen peroxide in its intercellular fluid than var. alicante. These data suggest that the inherent and increased resistance to Salmonella proliferation in var. cerasiforme compared to var. alicante is at least in part due to its increased levels of hydrogen peroxide in its intercellular fluid

Análisis multivariado aplicado a la representación de datos sintéticos de secuenciación de ARN

Tesis (Maestría en Estadística Aplicada) -- Universidad Nacional de Córdoba. Facultad de Ciencias Económicas. Escuela de Graduados; Argentina, 2017.La secuenciación de alto rendimiento de ARN genera grandes bases de datos con información que puede ser utilizada con diferentes objetivos. Una de las aplicaciones más utilizada consiste en resumir las lecturas de las secuencias agregándolas en función de una unidad de interés tal como gen, exón o transcript . En este tipo de análisis se obtienen matrices con datos de conteos correspondientes a cada individuo en estudio (filas) y asignados a una particular unidad de interés (columnas). En general el número de individuos es muy pequeño en relación al número de variables y los conteos presentan un rango de dispersión muy amplio. En esta tesis se comparan técnicas de análisis multivariado exploratorio a 2 y 3 vías de clasificación que contemplan la naturaleza de los datos obtenidos en experimentos de secuenciación de ARN. Utilizando datos sintéticos generados con la técnica de plasmodios se comparan transformaciones a los datos y medidas de disimilaridad empleadas en el análisis de cluster jerárquico, análisis de escalamiento multidimensional métrico y no métrico y en el análisis factorial multiple. La transformación de los conteos originales a través de funciones que utilizan logaritmo o el uso de disimilaridades basadas en correlacion de Spearman o disimilaridad Poisson rescata la estructura natural de las muestras en todos los métodos de análisis utilizados. La mera estandarización o normalización de los conteos no genera representaciones confiables. La elección de la mejor medida debe considerar el nivel de relación señal-ruido ya que no todas las medidas muestran la configuración natural de la muestras en función de la cantidad de transcripts expresados o no diferencialmente. Este aspecto debe considerarse al momento de representar las muestras utilizando todos transcripts obtenidos o filtrando por expresión diferencial.Fil: Reeb, Pablo Daniel. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas. Escuela de Graduados; Argentina

Wildlife in an anthropogenically-driven world: how humans have shaped the distribution, genetic composition, and gene expression of North American forest hawks (Genus: Accipiter)

Humans are causing drastic environmental change on a global scale and this trend strongly influences the evolution of species. It is also becoming clear that tolerances to anthropogenic disturbance varies widely among organisms. Therefore, understanding the mechanisms by which wildlife cope with humans is a pressing question in modern ecology. North America\u27s forest raptors (Genus: Accipiter) are a useful model for investigating the effects of humans on wildlife species. All three Accipiter species experienced historic demographic declines as a result of anthropogenic activities, yet each species has rebounded differently since these declines. One species in particular is now exploiting urban areas, despite the fact that all of these species were traditionally considered highly dependent on large contiguous forests for survival. This dissertation consists of one introductory chapter, three chapters involving research to improve our current understanding of the impacts of anthropogenic activities on the raptors, and two chapters focused on the development of tools for improving future avian research. The first chapter provides background information on the history of Accipiter hawks in the eastern United States. There are also basic descriptions of some of the novel genetic tools that are becoming increasingly valuable in this and other wildlife studies. In addition, this chapter provides justification for the research and an outline of the project goals. For the second chapter, I developed a spatial habitat model using Maximum Entropy to locate nesting habitat for northern goshawks (Accipiter gentilis) in New York State, a potential stronghold for this species in the east. This species is the most secretive of the Accipiters, considered highly sensitive to human disturbance, and a species of concern in many eastern states. The model predicted nesting habitat with high success (AUC = 0.87), and ground-truthing efforts identified two previously unknown nest territories. In addition, my model provides some evidence of a shift in forest cover preference by goshawks nesting in New York, as coniferous land cover was the most important predictor in the model (67%). Future modeling efforts should include additional and more detailed environmental input layers. In the third chapter, I developed a new mechanical lure owl for trapping nesting raptors that exhibited both realistic head and wing movements. The mechanical owl was tested on six species of raptors and capture rates were similar or better than previously reported with a live lure owl for five of the six species. In addition, average time to capture was eight minutes faster with the mechanical owl as compared to a live owl when trapping northern goshawks (p \u3c 0.01). A mechanical owl costs less and is ethically more appropriate to live lure owls and thus, the use of this type of owl may be warranted in future raptor research. For the fourth chapter, I investigated the genetic consequences of demographic declines in Accipiter hawks. I used microsatellite markers to test for evidence of significant genetic bottlenecks in northern goshawks and Cooper\u27s hawks (A. cooperii) in the northeastern United States. There was some evidence to suggest a bottleneck in goshawks using the heterozygosity excess method, while the M ratio method suggested a bottleneck in Cooper\u27s hawks. However, similar to previous studies, I found that the results of bottleneck testing are strongly dependent on mutation model parameters, which are not available for Accipiter hawks and numerous other non-model organisms. Still, by using the results from tests on both species, I was able to ascertain useful information about the relative impact of historic declines. The Cooper\u27s hawk likely experienced more drastic declines than goshawks, while the goshawk population has likely been small for a relatively long time. Finally, useful baseline information about the contemporary genetic structure of both species was gained from this research. There is no evidence of inbreeding in either population and both species have high levels of gene flow in the northeastern United States. In the fifth chapter, I compared two commercially available buffers for stabilization of RNA from avian blood for downstream RNA processing. Avian blood presents a particular challenge because it contains nucleated red blood cells and most buffers have been developed for blood with non-nucleated red blood cells (e.g. mammalian blood). Each buffer was subjected to a variety of room temperature incubation periods and freeze treatments, to simulate different field sampling scenarios. RNAlater outperformed RNAProtect; RNAlater reliably stabilized RNA regardless of treatment. However, RNA integrity numbers (RIN) varied widely between samples (1.7 -- 7.5). RNA from Cooper\u27s hawk blood stored in RNAlater was sequenced and mapped to the golden eagle (Aquila chrysaetos) genome. Quality assessment suggested that reads were of high quality regardless of RIN value. However, reads that aligned to the reference genome had relatively low sensitivity (\u3c14%) and a wide range of precision (10-61%). These results suggest that RNAlater can be used to obtain usable RNA for avian blood, but future research may be useful for improving stabilization buffers for species with nucleated red blood cells. The sixth and final chapter focuses on the Cooper\u27s hawk in urban environments. This species has recently been found nesting in high densities in urban centers and an extensive body of research has demonstrated differences between urban and exurban individuals. When colonizing urban areas, organisms can either adapt through heritable genetic mechanisms or acclimate through plastic mechanisms such as gene expression. Previous research suggested that highly mobile species may be more likely to acclimate since they are capable of moving away from potential stressors. Therefore, I used RNA-sequencing to compare gene expression patterns in the blood of urban and exurban adult and fledgling Cooper\u27s hawks in the Albuquerque, NM area. I also tested all individuals for the presence of an urban-associated parasite (Trichomonas gallinae). I found one and thirteen differentially expressed (DE) transcripts between urban and exurban adults and fledglings, respectively (q \u3c 0.05). For fledglings, more abundant transcripts in the urban environment were mostly associated with nucleotide processing, while those in exurban environments were mostly associated with immune response. The single transcript identified as DE in the adults was more abundant in urban environments and is associated with nucleotide processing, metal ion binding, and platelet production in humans. The greater number of DE transcripts in the fledglings may suggest that changes in gene expression may be especially important for the sedentary offspring of a highly mobile avian urban exploiter. In addition, six fledglings tested positive for Trichomonas spp.; three in each environment. Yet, immune related transcripts were expressed in much higher levels in all exurban individuals, regardless of parasite presence. Future research is warranted to determine if toxin loading in urban environments may lead to immunosuppression of offspring and potentially explain previously described mortality in urban nestlings from trichomoniasis infection