No description available.

The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers — fragments of the invading nucleic acids — into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving no time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, I show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. I also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. While the acquisition of CRISPR spacers is not random and while the supplementation of eDNA influences that bias, eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection. / Thesis / Doctor of Philosophy (PhD) / Phages are viruses that can infect and kill bacteria with a 99.9999% success rate. To defend themselves, bacteria have evolved an adaptive immune system called the CRISPR-Cas system. This system uses a piece of DNA, called a spacer, that matches the phage to destroy it. However, in order to use their CRISPR-Cas system, they need to obtain this spacer. Given how dangerous phages are, how bacteria acquire this spacer is a mystery. My project investigates the possibility that bacteria use DNA floating in the environment to vaccinate themselves against phages before ever encountering them. In this thesis I show that DNA floating in the environment helps bacteria acquire these spacers. I also show that it is specific sections of phage DNA that helps bacteria. This shows that bacteria can use their environment to defend themselves against threats before they even happen.

Utilizing environmental DNA (eDNA) for the detection of species in the field is a potentially cost-effective and time-saving technology that may be useful in understanding the distribution and abundance of threatened or endangered species such as the Eastern Massasauga (Sistrurus catenatus). I describe the development of an eDNA assay for the species and evaluate its ability to detect eDNA in laboratory and field conditions. In the field samples, I also investigated the potential for abiotic conditions to influence eDNA detection. Species-specific primers and probe were designed to amplify a 152 bp segment of the massasauga COI gene. Target eDNA could be detected in samples containing as little as 100 copies of target DNA/μL. Water samples collected from laboratory housed snakes indicated that eDNA can be detected in water 56 days after massasauga removal. Field samples were taken from crayfish burrows, known overwintering habitat for the species, from four sites that vary in snake use as ascertained by traditional visual surveys. Of the 60 burrows sampled, seven had a positive detection for massasauga eDNA with no difference in detection rate between DNA extracted from burrow water and burrow sediment. Occupancy models fitted to burrow water indicated that larger amounts of total DNA in a sample may increase the probability of detection of a massasauga eDNA. Large confidence intervals in site occupancy (ѱ) and burrow detection (Θ) values suggest that a larger sample size is needed for more reliable occupancy models. Abiotic conditions within crayfish burrows varied among sites but correlation with eDNA detection was not supported. Estimates of qPCR detection within a burrow with eDNA (ρ) suggest that up to 10 qPCR replicates per burrow sample may be necessary. Further studies need to examine eDNA degradation in the field, improve upon the limit of detection, and sample a larger number of sites for eDNA sampling to be a stand-alone survey method for Eastern Massasaugas.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 213-220).

Les polérovirus infectent de nombreuses plantes d’intérêt économique telles que la pomme de terre, la betterave à sucre et les cucurbitacées. Ces virus icosaédriques renferment un ARN simple brin et leur capside est constituée d’une protéine majeure (CP) et d’un composant mineur (RT*) localisé à la surface des virions. Ces virus sont restreints aux cellules du phloème dans lesquelles ils se multiplient et se déplacent. Les protéines CP et RT sont essentielles à la dissémination du virus par le puceron vecteur et à son mouvement dans la plante. L’objectif de cette étude a consisté à identifier dans les cellules du phloème, les protéines associées aux virions susceptibles d’intervenir dans le cycle viral en criblant une banque d’ADNc de cellules compagnes (CC) d’A. thaliana avec les protéines de structure ou des domaines protéiques du CABYV. Quatre gènes codant pour une protéine Heat Shock (HSP), la profiline 3 (PRF3) une glysosyl hydrolase ; et la protéine « Response to low sulfur 3 » ont été identifiés. Tous ces gènes candidats interagissent avec le domaine RTC-ter du CABYV et avec la protéine RT* pour la protéine HSP. En plus de ces gènes candidats, je me suis intéressée à la protéine ALY, identifiée au laboratoire, au cours du criblage d’une banque d’ADNc de puceron entier avec les deux protéines de structure du Turnip yellows virus (un autre polérovirus). Cette protéine possède quatre orthologues chez Arabidopsis susceptibles d’être impliquées dans le mécanisme de gene silencing mis en place contre le Tomato Bushy Stunt Virus. Les protéines ALY sont donc des candidats intéressants et j’ai montré une interaction entre les protéines de structure du CABYV et du TuYV et les quatre orthologues d’Arabidopsis. L’implication de ces gènes candidats n’a pas pu être confirmée à ce jour dans des mutants knock-out d’arabidopsis. Les résultats complexes obtenus pour le candidat PRF3 au cours des analyses de validation fonctionnelle, m’a conduit à étudier l’interaction entre ce candidat et le domaine RTC-ter du CABYV in planta par FLIM mais aucune interaction n’a pu être confirmée à ce jour. Tous les candidats isolés lors du criblage de la banque d’ADNc de CC interagissant avec le domaine RTC-ter du CABYV, ce travail m’a conduit à analyser le rôle dans le cycle viral de ce domaine et de la protéine RT (sous sa forme complète ou dépourvue du domaine RTC-ter), en étudiant l’accumulation de ces mutants dans les plantes et le clivage de la protéine RT. Tout d’abord, afin de localiser précisément le site de clivage de la protéine RT, des mutants ponctuels dans la zone de clivage ont été réalisés ce qui a permis de montrer que la structure secondaire de la protéine est importante pour son clivage. Puis, afin d’analyser le rôle du domaine RTC-ter dans le cycle viral, j’ai obtenu par délétion, un mutant n’exprimant plus ce domaine. Ce mutant synthétise uniquement la protéine RT tronquée, forme des particules virales semblables au virus sauvage et est transmissible par puceron. Par contre, de façon surprenante, ce mutant est incapable d’envahir les feuilles non-inoculées d’une plante. Ce résultat suggère que les deux formes de la protéine RT (complète et tronquée) sont indispensables au mouvement à longue distance du virus et nous proposons un modèle dans lequel le domaine C-terminal de la protéine RT agit en trans sur la particule virale pour promouvoir le mouvement du CABYV à longue distance. / Poleroviruses infect a wide range of cultivated plants such as potatoes, sugar beet and plants of Cucurbitaceae family. These viruses are restricted to phloem tissue where they replicate in nucleated cells and translocate over long distances through sieve elements. Polerovirus capsid is composed of the major coat protein (CP) and of a minor component referred to as the readthrough (RT*) protein and exposed at the outside of the particles. CP and RT proteins are essential for virus movement and transmission by aphids. The aim of this study is to identify phloem proteins interacting with viral proteins and potentially involved in viral cycle, by screening an A. thaliana companion cell (CC) cDNA library with structural proteins or protein domains of CABYV. Four genes encoding for a heat shock protein (HSP), a profilin (PRF3), a glycosyl hydrolase and the protein ”Response to low sulfur ” (LSU3) were identified and interact with the C-terminal part of the RT protein (RTC‑ter) and with the RT* protein for the HSP. An additional gene encoding for the protein ALY, identified in the laboratory, by screening an aphid cDNA library with structural proteins of the Turnip yellows virus (another polerovirus) was studied. This protein has four orthologues in Arabidopsis, involved in the gene silencing mechanism against Tomato Bushy Stunt Virus. Here we show that CABYV and TuYV structural proteins interact with the four orthologues of Arabidopsis. Involvement of these candidate genes was not confirmed in Arabidopsis knock-out mutants. In functional experiments, ambiguous results were obtained with PRF3 arabidopsis mutants, and this lead me to study the interaction between PRF3 protein CABYV RT c-ter domain by FLIM, but no interaction was found so far. As all candidat interact with the RTC-ter domain, we studied more precisely the role of this domain in the viral cycle and the role of the complete RT protein. We studied the in vivo RT protein processing and its consequences on systemic movement of CABYV mutants. Using a collection of point mutations introduced in the central domain of the CABYV RT protein, we approached the site of the RT processing and proposed that this process is affected by the secondary structure around the cleavage site. We also reported for the first time the generation of a polerovirus mutant able to synthesize only the RT* protein and to incorporate it into the particle. This mutant was unable to move systemically. Conversely another mutant producing a full-length RT protein impaired in correct processing and incorporating a shorter version of the RT* protein showed very weak systemic infection. These data are strongly in favor of a role of both RT proteins in efficient CABYV movement. An inefficient virus transport was still maintained in the absence of RT proteins suggesting an RT-independent movement pathway. Based on these results, we propose a model for CABYV long-distance transport in which the complete RT protein, or its C-terminal part, acts in trans on wild-type virions to promote their efficient long-distance transport.

Conservation of elasmobranch populations is often inhibited by a lack of data, particularly in understudied regions like the Red Sea. Survey efforts in this region have been infrequent and often highly localized. Establishing a broad baseline for elasmobranch diversity and abundance along the Saudi Arabian Red Sea coast could inform both conservation efforts and a nascent ecotourism industry. In this thesis, I describe a pilot study comparing biodiversity data from baited remote underwater video stations (BRUVS), unoccupied aerial vehicle surveys (UAVs), and eDNA sequencing at five islands in the Farasan Banks region of the Saudi Arabian Red Sea. Estimates of relative abundance were also compared between the BRUVS and UAVs. Each method identified species missed by the other two, but all three techniques exhibited clear habitat- and taxa-specific biases. I was able to identify key concerns for each approach that need to be addressed before large-scale implementation. If carefully planned and executed well, a full assessment of the Saudi Arabian coastline could establish a true baseline for shallow water elasmobranchs in the eastern Red Sea. Informing best conservation practices and identifying potential ecological attractions in accordance the environmental and economic goals of Saudi Arabia’s Vision 2030.

No description available.

Human disruption of climate, habitat, and ignition has altered the behavior of wildland fire at local to continental scales. In many regions, novel fire regimes are emerging that threaten to exceed the capacity for local management to protect human wellbeing and ecosystem function. Simultaneous changes in climate, species composition, and fire management have resulted in extreme fire behavior in many regions. For the Western United States, the emerging novel fire regime consists of more frequent, severe, and intense wildfires, with annual area burned by wildfire having doubled and high-severity wildfire area having increased 8-fold since the 1980s. The impacts of these increasing stresses in the Great Basin is especially pressing when combined with the many years of historically poor resource management. Here we complete a literature review of changing wildfire regimes globally (chapter 1) and a study of how the abiotic and biotic aspects of aquatic ecosystems stabilize after a megafire in the western United States (chapter 2).

<p>  </p> <p>Semi-aquatic mammals, such as American beavers (<em>Castor canadensis</em>), muskrats (<em>Ondatra zibethicus</em>), North American river otters (<em>Lontra canadensis</em>), and American mink (<em>Neogale</em> <em>vison</em>), often play important roles in their ecosystem. Beavers and muskrats can manipulate plant community structure through the use of woody debris and forbs. As mesocarnivores, North American river otters and American mink can also drive community structure through the predation. Traditionally, these species are monitored using sign surveys (i.e., walking transects and visually identifying scat, tracks, and latrines). Camera trapping has also been used to survey semi-aquatic species occupancy to a lesser extent. However, due to their almost exclusive use of edge habitat, they may be ideal species to camera trap. Another more recently employed survey method is environmental DNA (eDNA), which involves the extraction of DNA from environmental samples (such as soil, water, air, and snow) to determine species occupancy. In this study, I evaluate environmental DNA and camera trapping as survey methods for detecting semi-aquatic mammals around northeastern Indiana. In the first chapter, I used eDNA sampling and camera trapping to monitor seven sites for three weeks during March – May 2021 in order to determine the presence of American mink. I found that the naïve occupancy for each site was 0.86. Although the detection probability of eDNA was lower than that of camera trapping (0.25 and 0.36, respectively), the occupancy models created suggest that there was no difference in detection probability between the two methods. I also compared the cost and time spent per sample and found that both were 20% lower for eDNA than camera trapping. The results of my study suggest eDNA may be a cost- and time-effective method for surveying for American mink occupancy. The objective of my second chapter was to determine the number of camera traps required to obtain reliable data for detecting semi-aquatic mammals. A minimum requirement for number of camera traps would be useful knowledge for wildlife managers in terms of budgeting and resource management and could also help to refine current camera trapping methodologies. I camera trapped four ponds for four weeks during June – July 2021, varying the number of camera traps (1 – 5) used at each pond each week. I collected a total of 66,543 photos and detected one semi-aquatic mammal throughout the study period (<em>Neogale vison</em>). Due to the lack of semi-aquatic mammals detected, I could not perform any analyses.</p>

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23959号 / 農博第2508号 / 新制||農||1092(附属図書館) / 学位論文||R4||N5394(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 益田 玲爾, 教授 三田村 啓理, 准教授 甲斐 嘉晃 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM