Since the 1980’s, there has been an unprecedented decline in the reef-building Caribbean corals, Acropora cervicornis and A. palmata, which has led to their listing as “threatened” under the U.S Endangered Species Act. Despite this protective status, these Acropora species continue to experience declines primarily attributed to disease, global climate change, and storm damage. Recent evidence suggests the hybrid of these threatened species (A. prolifera) is found at abundances similar to or higher than the parental species at many sites throughout the Caribbean. However, there is still much that is unknown as to how and why hybrids may be increasing in abundance at select sites. In 2007, scientists from NOAA NMFS established 9 permanent transects at three sites in the USVI to quantify fish diversity and coral tissue condition in A. cervicornis thickets. Over the years, they observed that A. prolifera seemed to be increasing in abundance on transects that were once dominated by A. cervicornis. This dataset provided a unique opportunity to investigate whether a shift from a threatened parental species to its hybrid may have occurred. This study has two objectives, (1) to quantify the change in A. cervicornis and A. prolifera percent cover and colony health over a 9-year period, and (2) to compare the genotypic diversity among the three Caribbean acroporids on and near the transects to determine the primary method of propagation, i.e., sexual versus asexual. For this study, I used transect photographs taken in March, July and November 2009, April 2012, and August 2017 to compare intra- and interannual variation in acroporid cover and colony health. Striking losses were observed in A. cervicornis cover between March 2009 and August 2017. At Thatch Cay, A. cervicornis declined from 25.7% to 8.9% between March 2009 and November 2009, but remained stable (10.2%) up to August 2017. Acropora cervicornis cover declined from 13.2% to 0% at Lovango Cay, and from 8.2% to 0% at No-Name Bay. At the one site (No-Name Bay) that A. prolifera was present during the original surveys of the transects, the percent cover remained relatively high and stable over the sample period. At No-Name Bay, A. prolifera percent cover (18.2%) was significantly higher than A. cervicornis (5.4%) by November 2009. It appears that A. prolifera expanded in the habitat left void by the decline in A. cervicornis. The general health of A. cervicornis based on the amount of healthy versus white and pale tissue appeared to decline at all sites between March 2009 and November 2009. To determine if the high percent cover on some transects was derived from asexual propagation or sexual recruitment, 139 tissue samples were collected in 2017 and genotyped using five microsatellite markers. No significant difference in genotypic richness (number of unique genotypes divided by the sample size) was observed among A. cervicornis (0.62), A. prolifera (0.64), and A. palmata (0.68). This suggests that the hybrid colonization is from multiple sexually derived individuals, not just asexual propagation from a rare hybridization event. High genotypic diversity, stable population abundance, and healthier colonies, suggest acroporid hybrids may become the primary habitat building coral of shallow reefs in the U.S. Virgin Islands. Due to considerable differences in morphologies between A. cervicornis and A. prolifera, it is unclear how a shift to the hybrid may affect the organisms that occupy acroporid structure and if the same ecological functions can be fulfilled.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20953号 / 理博第4405号 / 新制||理||1633(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 木庭 啓介, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Las plantas acuáticas del género Ruppia habitan lagunas costeras, salinas, humedales y aguas salinas interiores, jugando un papel ecológico clave. Estos sistemas se caracterizan por variaciones extremas de las condiciones ambientales tales como salinidad, temperatura e inundación. La compleja historia evolutiva del género Ruppia ha dificultado la delimitación de especies en el género. Estas especies han desarrollado una gran diversidad de estrategias biológicas para sobrevivir en estos ambientes extremos, tales como ciclos de vida anuales y perennes, reproducción sexual y propagación vegetativa, estrategias de polinización epihidrófila e hidroautogámica, así como autofecundación y fecundación cruzada. Además, existen diferentes vectores que pueden facilitar el flujo de genes entre las poblaciones, incluyendo corrientes marinas, aves acuáticas y peces. Estas características biológicas de Ruppia tienen una influencia importante en sus patrones de especiación, diversidad genotípica y genética, y su estructura poblacional. Por lo tanto, los análisis genéticos proporcionan información importante para delimitar especies y taxones dentro de este género, evaluar la diversidad e identificar procesos y flujos que actúan a distintas escalas temporales y espaciales. Los objetivos principales de esta tesis son: inferir los procesos evolutivos y biológicos de especiación y diversificación, y evaluar la prevalencia y estructura poblacional de especies Europeas del género Ruppia. Esta tesis se centra principalmente en las zonas costeras de la Península Ibérica, debido a la alta diversidad de especies de Ruppia registradas en esta zona geográfica, y al hecho de que en muchas ocasiones es posible encontrar poblaciones de diferentes especies en simpatría. Para alcanzar estos objetivos, en el Capítulo I, se desarrollaron y validaron diez nuevos marcadores moleculares polimórficos (es decir, microsatélites) para Ruppia cirrhosa. Adicionalmente se obtuvo amplificación cruzada con otros dos microsatélites descritos anteriormente para R. maritima. Estas herramientas moleculares son importantes para el estudio de plantas clonales y se han utilizado junto con secuencias nucleares y del cloroplasto en los siguientes capítulos. En el Capítulo II, se estudió la sistemática del género Ruppia en Iberia considerando criterios morfológicos, marcadores nucleares altamente variables (microsatélites) y secuencias nucleares (ITS) y del cloroplasto (psbA-trnH). Al realizar la filogenia utilizando marcadores con diferentes tiempos de mutación y mecanismos hereditarios, pudo identificarse el importante papel de la hibridación y la introgresión en la historia evolutiva de este género. De las tres especies tradicionalmente descritas en la Península Ibérica, se observó que R. drepanensis y R. cirrhosa se situaron en el mismo clado filogenético tanto para los marcadores nucleares como para los cloroplastos, por lo que pueden considerarse especies hermanas. R. maritima está incluida en un clado más distante filogenéticamente, apoyado por ambos marcadores. Además, dos nuevas entidades genéticas fueron identificadas, R. cf. maritima y "R. híbrido", las cuales mostraron algunas incongruencias entre los árboles filogenéticos del núcleo y del cloroplasto, así como una combinación de alelos de microsatélites que sugieren la existencia de efectos de hibridación y/o introgresión. En el Capítulo III, mediante el estudio de microsatélites en diferentes poblaciones de R. cirrhosa de la Península Ibérica y Sicilia, se detectó una fuerte estructura genética poblacional. En términos generales, se registró un bajo nivel de flujo génico, el cual fue más importante entre poblaciones geográficamente cercanas o ubicadas en el mismo cuerpo hidrológico. Además, se evaluaron diferentes hipótesis para explicar la conectividad entre las poblaciones a través de correlaciones entre distancias geográficas y genéticas, sugiriendo que el vector de dispersión más probable entre las poblaciones de R. cirrhosa en la Península Ibérica son las aves acuáticas. Al compilar los resultados del Capítulo II y el Capítulo III, se evaluaron los efectos de diferentes estrategias reproductivas sobre la diversidad genotípica y genética de Ruppia. Todas las entidades genéticas mostraron elevadas tasas de reproducción sexual. En R. cirrhosa, los mayores índices de reproducción sexual se detectaron en los hábitats más inestables hidrológicamente. Estas perturbaciones podrían promover la germinación y el establecimiento de semillas por una baja competencia interespecífica por el espacio, la luz y otros recursos existentes en praderas menos densas. Los mayores valores de diversidad genética detectados en los epihidrófilos R. drepanensis, R. cirrhosa y probablemente en “R. híbrido” (ésto no se ha confirmado) que en la hidroautogámica Ruppia cf. maritima, sugieren una fuerte influencia del modelo de polinización sobre los patrones de diversidad genética. En el Capítulo IV, R. maritima fue identificada por primera vez en Cabo Verde (Isla de Santiago) a partir de análisis morfológicos y filogenéticos. Esta información amplía la distribución geográfica de esta especie al África Occidental.

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs – Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.

Epigenetic regulatory mechanisms play important roles in the control of lineage-specific differentiation during development. However, mechanisms that regulate higher-order chromatin remodelling and transcription of keratinocyte-specific genes that are clustered in the genome into three distinct loci (Keratin type I/II loci and Epidermal Differentiation Complex (EDC) during differentiation of the epidermis are poorly understood. By using 3D-Fluorescent In Situ Hybridization (FISH), we determined that in the epidermal keratinocytes, the KtyII and EDC loci are located closely to each other in the nuclear compartment enriched by the nuclear speckles. However, in KtyII locus knockout mice, EDC locus moved away from the KtyII locus flanking regions and nuclear speckles towards the nuclear periphery, which is associated with marked changes in gene expression described previously. Chromatin architectural protein Ctcf has previously been implicated in the control of long-range enhancer-promoter contacts and inter-chromosomal interactions. Ctcf is broadly expressed in the skin including epidermal keratinocytes and hair follicles. Conditional Keratin 14-driven Ctcf ablation in mice results in the increase of the epidermal thickness, proliferation, alterations of the epidermal barrier and the development of epidermal pro-inflammatory response. Epidermal barrier defects in Krt14CreER/Ctcf fl/fl mice are associated with marked changes in gene expression in the EDC and KtyII loci, which become topologically segregated in the nucleus upon Ctcf ablation. Therefore, these data suggest that Ctcf serves as critical determinant regulating higher-order chromatin organization in lineage-specific gene loci in epidermal keratinocytes, which is required for the proper control of gene expression, maintenance of the epidermal barrier and its function.

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