In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic understanding of emerging model organisms in the past, but advancing high-throughput RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq neglects essential considerations for transcriptomic studies including the spatial and temporal expression of transcripts, available budget, experimental design needs, and validation of data. A synergy of spatiotemporal transcriptomic techniques is needed, using the bulk and unbiased analysis of RNA-seq and the visual validation and spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our molecular understanding of non-model organisms and establish the background data needed for advancing research techniques. A prime example lies within an emerging model of the marine science and symbiosis fields, where I present a case study on a threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will help future studies reveal the gene regulation underpinning the establishment, persistence, and breakdown of this complex symbiotic relationship.

The relationship between international commerce and sustainable development is the subject of much controversy that is exacerbated by geographic boundaries that are co-jointly developed, shaped, and sustained by regional trade agreements. The outcomes of three Americas trade agreements - NAFTA, CAFTA, and USPTPA - are analyzed across three dimensions: economic, ecological, and labor. The three dimensions collectively form for each trade region a specific ECOL niche that is concurrently subject to national variation. We propose and find that low-ECOL niches in the Americas appear to attract more foreign trade with investment. Nevertheless, this tentative finding seems not to hold for those corporations that seek out strong ECOL niche countries like Costa Rica.

In collaboration with Afry, the focus of this thesis is to facilitate the transition from an Industrial park into an Eco-park using industrial symbiosis strategy, in the industrial harbor of Malmö. To transition into a collaboration where physical exchanges of energy, waste, materials and other by-products between different facilities takes place. Some of the keys to an industrial symbiosis are proximity, government planning and self-organization. The aim of the thesis is to create a digital platform that can facilitate the connectivity between facilities and organizations and to inspire a social transition. Therefore, this thesis uses transition design as an approach, touching upon topics such as organizational collaboration, industrial symbiosis strategy and knowledge sharing. Whilst using Research through design for designing a technology that can inspire collective transition into a more sustainable future. Design methodology from Service design and Interaction design, such as user researching, customer journey mapping and prototyping, is used when designing for a somewhat unexplored area in Sweden.

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.

Corals are cnidarian animals that build the founding structures of tropical reefs, which survival depends upon the obligate symbiotic association to photosynthetic dinoflagellate algae in the family Symbiodiniaceae. As corals are facing increasing environmental and anthropogenic stress, understanding the molecular principles governing this unique symbiotic association is crucial to predict their adaptive potential. Due to logistic, costly, and experimental difficulties of working with corals, we use the sea anemone Aiptasia (sensu Exaiptasia pallida) as a tractable model organism for the molecular study of cnidarian-algal symbiosis. A major advantage of Aiptasia is that it establishes a facultative symbiotic association with Symbiodiniaceae algae, that is, this anemone can be maintained in an aposymbiotic (symbiont-free) state, allowing for comparison of symbiotic and ‘control’ aposymbiotic processes. The main aim of this dissertation was to investigate the signaling pathways involved in the regulation of this symbiotic interaction, and in particular, phosphorylation-mediated protein signaling. Phosphorylation is indeed a major post-translational modification that mediates signal transduction within and across cells. To investigate if protein phosphorylation regulates the complex intercellular signaling that occurs between symbiotic partners, a mass spectrometry-based phosphoproteomic approach was employed. Given the novelty of this application in the field of coral reef biology, the first research chapter details the development and optimization of a protocol that allows quantification of protein phosphorylation in the sea anemone Aiptasia. This chapter includes mass spectrometric analysis in 1) data-dependent acquisition (or shotgun proteomics) for the generation of a so-called assay (spectral) library, a reference dataset that servers for 2) accurate and reproducible label-free quantification of protein phosphorylation in data-independent acquisition (DIA/SWATH-MS). In the second research chapter, the developed protocol was employed to generate a phosphopeptide assay (spectral) library for aposymbiotic and symbiotic Aiptasia, which would allow further quantification of protein phosphorylation across symbiotic conditions. We consistently quantified more than 3,000 phosphopeptides, totaling more than 1,600 phosphoproteins, across biological replicates and symbiotic conditions. Characteristic phosphoproteomic profile distinguished the two symbiotic groups and differential phosphorylation targeted biological processes that have not been previously described in the context of cnidarian-algal symbiosis, namely the phospholipase D signaling pathway and protein processing in the endoplasmic reticulum. We suggest that changes in the phosphorylation status of these signaling pathways may have a potentially relevant role in the control of an established cnidarian-algal association.

L’insecte phytophage Riptortus pedestris, appartenant au sous-ordre des Hétéroptères, est un ravageur notoire de cultures agricoles en Asie du sud-est qui se nourrit préférentiellement de plants de soja. Cette punaise est associée à une bactérie symbiotique du genre Burkholderia nommée Burkholderia insecticola, localisée dans une région spécifique de l’intestin de l’insecte appelée la région M4. Cette région M4, organisée en cryptes, constitue l’organe symbiotique dans lequel le symbiote prolifère de manière extracellulaire. Cette interaction favorise la croissance et le développement de la punaise. Récemment, il a été montré que Riptortus produit des peptides antimicrobiens au sein des cryptes, appelés “crypt-specific cysteine-rich peptides” ou peptides CCR pour lesquels le symbiote est particulièrement résistant. Il a été proposé que les peptides antimicrobiens de l’hôte,incluant les peptides CCR, participent à la colonisation spécifique de l’organe symbiotique par B. insecticola. Dans ce travail, une approche Tn-seq a été utilisée pour identifier les gènes bactériens impliqués dans la résistance aux peptides antimicrobiens et dans la symbiose. Dans un premier temps, la robustesse de la méthode Tn-seq a été évaluée en identifiant le génome essentiel de B. insecticola. Puis dans un second temps, les facteurs bactériens impliqués dans la résistance aux peptides antimicrobiens ont été caractérisés via une approche gènes-candidats et l’approche Tn-seq. Dans une dernière partie, une expérience de Tn-seq in vivo a permis d’évaluer l’ampleur du goulot d’étranglement sur la population symbiotique lors de l’infection de l’organe symbiotique et d’identifier les facteurs symbiotiques impliqués dans la colonisation de R. pedestris. / The phytophagous insect Riptortus pedestris, belonging to the Heteroptera suborder, is a notorious crop pest in South-Eastern Asia which feeds preferentially on soybean plants. This bean bug is associated with a bacterial symbiont, a specific Burkholderia species named Burkholderia insecticola, located in the M4 region of the insect’s midgut. This M4 region is organized in crypts and constitutes the symbiotic organ where the symbiont proliferates extracellularly. This interaction promotes the growth and the development of the bean bug. Recently, it was demonstrated that Riptortus produces antimicrobial peptides in the midgut crypts called crypt-specific cysteine-rich peptides (CCR) for which the bacterial symbiont demonstrates a high resistance profile. It was proposed that host antimicrobial peptides, including the CCR peptides, contribute to the specific colonization of the symbiotic organ by B. insecticola. In this work, a Tn-seq approach was used to find bacterial fitness genes involved in antimicrobial peptide resistance and symbiosis. First, the robustness of the Tn-seq method was assessed by identifying the essential genome of B. insecticola. Second, the bacterial factors for antimicrobial peptide resistance were characterized, based on both a candidate-gene and the Tn-seq approach. Finally, a Tn-seq in vivo experiment was performed to reveal the infection bottleneck effect on the symbiotic population and to identify the bacterial symbiosis factors for the colonization of R. pedestris.

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

As data collection and modeling improve, ecologists increasingly discover that interspecies dynamics greatly affect the success of individual species. Models accounting for the dynamics of multiple species are becoming more important. In this work, we explore the relationship between mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) and two mutualistic fungi, Grosmannia clavigera and Ophiostoma montium. These species are involved in a multipartite symbiosis, critical to the survival of MPB, in which each species benefits. Extensive phenological modeling has been done to determine how temperature affects the timing of life events and cold-weather mortality of MPB. The fungi have also been closely studied to determine how they interact with MPB and how they differ in terms of virulence, response to temperature, and nutritional benefits to developing beetles. Overall, researchers consider G. clavigera to be the superior mutualist. Beetles developing near G. clavigera are larger, produce more brood, and have higher survival rates. Regarding temperature preferences, G. clavigera is considered “cool-loving,” growing at cooler temperatures than O. montium. These findings lead researchers to wonder 1) why has G. clavigera not displaced iv O. montium from the mutualism (if it is the superior mutualist) and 2) what will happen to the MPB-fungus mutualism in the face of a warming climate. In this work we present two models connecting fungal growth in a tree to predictions of MPB emergence: a stochastic, individual-based model and a deterministic, tree-based model. We begin by exploring whether variability in temperature can act as a stabilizing mechanism and find that temperature variability due to MPB periodically transitioning between different thermal environments is the most likely explanation for the continued presence of both fungi in the mutualism. Using the second model, we parameterize and validate the model using attack and emergence observations of MPB and the fungi they are carrying. In the process, we test several submodels to learn more about specific MPB-fungi interactions. Finally, utilizing information from previous fungal growth experiments, we test and parameterize several growth rate curves using Bayesian techniques to determine whether the inclusion of prior knowledge can lead to more realistic fits.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19070号 / 人博第723号 / 新制||人||173(附属図書館) / 26||人博||723(吉田南総合図書館) / 32021 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 市岡 孝朗, 教授 加藤 眞, 教授 瀬戸口 浩彰 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20442号 / 農博第2227号 / 新制||農||1050(附属図書館) / 学位論文||H29||N5063(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 澤山 茂樹, 教授 左子 芳彦, 准教授 中川 聡 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM