Several papers have reported on the development of antibiotic resistance and implications for human medicine but fewer deal with environmental impacts of antibiotic use. Marine sea cage aquaculture in SE Asia is often established close to coral reef ecosystems. Large amounts of antibiotics are used in the cultivation of fish and lobster and hence released directly into the environment. This study investigates the antibiotic practices in sea cage farms producing fish and spiny lobster in Vietnam, mainly for the domestic market. There are approximately 3500 sea cage farms in Vietnam and we performed semi-structured interviews with 109 sea cage farmers asking them if they use antibiotics and if so; what sort/when/how often/how much. We found that the Vietnamese cage farmers are using antibiotics in an unstructured way, which seems to have little or no effect on the survival of the stock, or profit of the farm. The fact that the farmers live at their farm and use the sea next to the cages both for fishing and collecting filter-feeding bivalves for direct consumption, as well as a toilet, poses an additional risk for the spreading of human antibiotic resistant pathogens. Thirteen different antibiotics were found in the study. Eighty-two percentage of the lobster farmers and 28% of the fishfarmers used antibiotics. The average amounts used were over 5 kg per produced ton of lobster and about 0.6 kg per ton of fish, which is much higher than in other studies. Several antibiotic substances listed as critical and highly important for human medicine by WHO were used prophylactically and routinely with little control and enforcement of regulations. We tested and detected antibiotic resistance to Tetracycline, Vancomycin and Rifampicin in the coral associated bacteria Bacillus niabensis as far as 660m from fish farms with resistance decreasing with distance from the cage farms. The antibiotics are likely to have negative effects on the coral-symbiont relationship adding further risks to an already stressed environment.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic Glade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular Glade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate i-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular Glade. Molecular clock analysis indicated that the intracellular Glade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

Among dinitrogen (N₂)-fixing some cyanobacteria can establish symbiosis with a broad range of host plants from all plant lineages including bryophytes, ferns, gymnosperms, and angiosperms. In the boreal forests, the symbiosis between epiphytic cyanobacteria and feathermosses Hylocomium splendens and Pleurozium schreberi is ecologically important. The main input of biological N to the boreal forests is through these cyanobacteria, and thus, they greatly contribute to the productivity of this ecosystem. Despite the ecological relevance of the feathermoss symbiosis, our knowledge about the establishment and maintenance of cyanobacterial-plant partnerships in general is limited, and particularly our understanding of the feathermoss symbiosis is rudimentary.

The first aim of this thesis was to gain insight on the genomic rearrangements that enabled cyanobacteria to form a symbiosis with feathermosses, and their genomic diversity and similarities with other plant-symbiotic cyanobacteria partnerships. Genomic comparison of the feathermoss isolates with the genomes of free-living cyanobacteria highlighted that functions such as chemotaxis and motility, the transport and metabolism of organic sulfur, and the uptake of phosphate and amino acids were enriched in the genome of plant-symbiotic cyanobacteria.

The second aim of this PhD study was to identify cyanobacterial molecular pathways involved in forming the feathermoss symbiosis and the regulatory rewiring needed to maintain it. Global transcriptional and post-transcriptional regulation in cyanobacteria during the early phase of establishment of the feathermoss symbiosis, and after colonization of the moss were investigated. The results revealed that the putative symbiotic gene repertoire includes pathways never before associated with cyanobacteria-plant symbioses, such as nitric-oxide sensing and regulation, and the transport and metabolism of aliphatic sulfonate.

The third aim was to explore the role of the cyanobacterial community in contributing to the temporal variability of N₂-fixation activity. Results from a field-study showed that temporal variation in N₂-fixation rates could be explained to a high degree by changes in cyanobacterial community composition and activity. In particular, the cyanobacteria belonging to the genus Stigonema - although not dominating the community- appeared to be the main contributors to the N₂-fixation activities. Based on this result, it is suggested that this genus is responsible for the main input of N in the boreal forest ecosystems.

The last aim was to understand how the relationship between cyanobacterial community composition and N₂-fixation activity will be affected by climatic changes such as, increased temperature (11^oC compared to 19^oC) and CO₂ level (500 ppm compared to 1000 ppm). Laboratory experiments highlighted that 30 weeks of combined elevation of temperature and CO₂ resulted in increased N₂-fixation activity and moss growth rates. The observed increases were suggested to be allocated to reduced cyanobacterial diversity and changes in community composition, resulting in the dominance of cyanobacteria adapted to the future abiotic condition.

Dinitrogen (N₂)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological N input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and incompetent Nostoc strains, using a proteogenomics approach and an experimental setup allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss-cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria-plant symbioses, with Nostoc retaining motility, and lacking modulation of N₂-fixation, photosynthesis, GS-GOGAT cycle, and heterocyst formation. The results expand our knowledgebase of plant-cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria-feathermoss symbiosis. 

Dinitrogen (N-2)-fixation by cyanobacteria living in symbiosis with pleurocarpous feather mosses (for example, Pleurozium schreberi and Hylocomium splendens) represents the main pathway of biological N input into N-depleted boreal forests. Little is known about the role of the cyanobacterial community in contributing to the observed temporal variability of N-2-fixation. Using specific nifH primers targeting four major cyanobacterial clusters and quantitative PCR, we investigated how community composition, abundance and nifH expression varied by moss species and over the growing seasons. We evaluated N-2-fixation rates across nine forest sites in June and September and explored the abundance and nifH expression of individual cyanobacterial clusters when N-2-fixation is highest. Our results showed temporal and host-dependent variations of cyanobacterial community composition, nifH gene abundance and expression. N2-fixation was higher in September than June for both moss species, explained by higher nifH gene expression of individual clusters rather than higher nifH gene abundance or differences in cyanobacterial community composition. In most cases, 'Stigonema cluster' made up less than 29% of the total cyanobacterial community, but accounted for the majority of nifH gene expression (82-94% of total nifH expression), irrespective of sampling date or moss species. Stepwise multiple regressions showed temporal variations in N-2-fixation being greatly explained by variations in nifH expression of the 'Stigonema cluster'. These results suggest that Stigonema is potentially the most influential N-2-fixer in symbiosis with boreal forest feather mosses.

In boreal forests, N₂-fixation by filamentous heterocystous cyanobacteria in symbiosis with pleurocarpous feathermosses represent the main biological input of new nitrogen (N), and greatly support the surrounding forest productivity. In these ecosystems, climate change is expected to result in 2-8°C increase in temperature and two times greater carbon dioxide (CO₂) over the next century, yet little is known about the effects of these factors on the community composition of cyanobacteria in symbiosis with feathermosses. In particular, it is unknown how N₂-fixation activity will change and the resulting impact on the moss growth rate (MGR). Here, we reconstructed the symbiosis between the common feathermoss Pleurozium schreberi and different cyanobacteria isolated from feathermosses. Feathermosses were inoculated with different strains of cyanobacteria and exposed to different temperature (11°C and 19°C) and CO₂ (500 ppm and 1000 ppm) conditions. Changes in MGR, N₂-fixation rate, and cyanobacterial community structure and composition were measured at distinct time points: 5, 10, 20, and 30 weeks of exposure. Our results indicate that both elevated temperature and CO₂ levels can individually and combined have positive effects on the N₂-fixation activities, and consequently on MGR. The underlying biotic mechanisms for increased N₂-fixation and MGR were changes in the cyanobacterial community diversity and composition.  In general, we observed a reduction in cyanobacterial diversity and an increase in cyanobacterial strain abundance that have adapted and dominated under the elevated temperature and CO₂ levels. 

The cyanobacteria belonging to the genus Nostoc comprise free-living strains but also facultative plant-symbionts. Symbiotic strains can enter into symbiosis with a taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here we compared the genomes derived from eleven symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, suggesting that this trait was most likely gained several times in the evolution of the Nostocales. Facultative symbiotic Nostoc strains showed enriched functions in the transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, amino acid and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains by a gain/enrichment of genes encoding proteins to generate L-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell-walls. These compounds could function as C sources for members of the intracellular clade. Molecular clock analysis suggested that the intracellular clade emerged ~600 million years ago, which would predate the origin of land plants. This suggest that intracellular cyanobacterial symbioses may have even predated the emergence of extant terrestrial plants.