The use of stable nitrogen isotope data in ecological and physiological studies is based in the assumption that nitrogen fractionates predictably during metabolism, leading to a broadly conserved pattern whereby consumers are isotopically enriched with respect to their diets. The application of stable isotope data to such studies is limited is by our understanding of the factors in that cause variability in the Δ15N values of consumers. In particular, parasites and fluid-feeders have been shown to demonstrate isotopic depletion with respect to their food sources. One factor that has been suggested to influence the Δ15N values seen in fluid-feeding consumers is the presence of endosymbionts and their contribution to nitrogen metabolism. The experiments described in this thesis directly test the hypothesis that the endosymbiotic bacteria Buchnera aphidicola is influencing the Δ15N value of the pea aphid on host alfalfa plants. Here I find that although aphids cured of their bacterial symbionts are less isotopically depleted than untreated aphids, they are still not enriched with respect to their phloem sap diet, indicating that endosymbiont metabolism alone is not responsible for the isotopic depletion observed in pea aphids. Metabolism of nitrogen in the pea aphid-Buchnera symbiosis has been well described with decades of physiological studies and with the publication of the pea aphid and Buchnera genomes. The two key features of metabolism in the pea aphid-Buchnera symbiosis are the recycling of waste ammonia by the aphid and the upgrading of the nonessential amino acids found in phloem sap to essential amino acids through collaborative metabolism between the pea aphid and Buchnera. Consistent with the described role of Buchnera in nitrogen metabolism, amino acid analyses of symbiotic and aposymbiotic aphids demonstrates an accumulation of the nonessential amino acids glutamine and glutamate and lower amounts of essential amino acids in the aposymbiotic aphids. I tested the influence of dietary amino acid profile on the Δ15N value of pea aphids and found that aphids are only isotopically depleted when they feed on diets with unbalanced amino acid compositions and are isotopically enriched when fed on a diet with a balanced profile of amino acids. I used isotopically labeled fructose to determine whether the difference in Δ15N value of pea aphids on diets of varying amino acid profiles is correlated to the amount of de novo amino acid synthesis occurring in the aphid. I found that there was a significantly higher incorporation of the labeled carbon backbone in the protein of pea aphids feeding on the unbalanced diets, supporting the idea that increased de novo amino acid synthesis are responsible for the differences in Δ15N values among aphids feeding on the two diets. The findings of this study highlight the influence of endosymbionts on the Δ15N values for pea aphids, demonstrate that dietary amino acid composition can influence the Δ15N value of pea aphids through the demand for metabolic upgrading of amino acids, and provide a model for the study of Δ15N values in systems where metabolism has been well characterized by experimental and genomic data.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Mark E. Warner, College of Marine and Earth Studies. Includes bibliographical references.

Die arbuskuläre Mykorrhiza ist die wahrscheinlich älteste Form der Wurzelsymbiosen zwischen Pflanzen und Pilzen und hat sich vor 420 Millionen Jahren entwickelt. In dieser Symbiose, die zwischen nahezu allen Landpflanzen und Pilzen des Reiches Glomeromycota ausgebildet wird, versorgt der Pilz die Pflanze mit Nährstoffen, wobei die verbesserte Versorgung mit Phosphat für die Pflanze sicher den größten Vorteil darstellt. Im Gegenzug erhält der Pilz Zucker, welche die Pflanze aus der Photosynthese bereitstellt. Zu hohe Phosphatkonzentrationen im Boden oder Dünger führen allerdings zu einer Verringerung in der Ausprägung der arbuskulären Mykorrhiza. Diese Unterdrückung der Symbiose wird nicht durch eine lokale Reaktion der Wurzeln ausgelöst, sondern in erster Linie durch einen hohen Phosphatgehalt im Pflanzenspross. Somit handelt es sich also um eine systemische, also dem Gesamtsystem „Pflanze“ betreffende Antwort. Die molekularen Mechanismen dieser Anpassung sind noch wenig bekannt und sind vor allem für die Agrarwirtschaft von besonderem Interesse. Eine Mikro-RNA (miRNA) des bereits bekannten Phosphathomöostasesignalwegs (PHR1-miRNA399-PHO2 Signalweg) akkumuliert verstärkt in mykorrhizierten Wurzeln. Das deutet daraufhin, dass dieser Signalweg und diese miRNA eine wichtige Rolle in der Regulation der arbuskulären Mykorrhiza spielen. Ziel dieser Studie war es neue Einblicke in die molekularen Mechanismen, die zur Unterdrückung der arbuskulären Mykorrhiza bei hohen Phosphatkonzentrationen führen, zu gewinnen. Dabei sollte der Einfluss von PHO2, sowie von miRNAs in dieser Symbiose genauer untersucht werden. Ein funktionelles Ortholog von PHO2, MtPho2, wurde in der Pflanze Medicago truncatula identifiziert. MtPho2-Mutanten, welche nicht mehr in der Lage waren ein funktionales PHO2 Protein zu exprimieren, zeigten schnellere Kolonisierung durch den AM-Pilz. Jedoch wurde auch in den mtpho2-Mutanten die Symbiose durch hohe Phosphatkonzentrationen unterdrückt. Dies bedeutet, dass PHO2 und somit der PHR1-miRNA399-PHO2 Signalweg eine wichtige Funktion während der fortschreitenden Kolonisierung der Wurzel durch den Pilz hat, aber und weitere Mechanismen in der Unterdückung der Symbiose bei hohen Phosphatkonzentrationen beteiligt sein müssen. Die Analyse von Transkriptionsprofilen von Spross- und Wurzeln mittels Microarrays zeigte, dass die Unterdrückung der AM Symbiose durch hohe Phosphatkonzentrationen möglicherweise auf eine Unterdrückung der Expression einer Reihe symbiosespezifischer Gene im Spross der Pflanze beruht. Um die Rolle weiterer miRNA in der AM Symbiose zu untersuchen, wurden mittels einer Hochdurchsatz-Sequenzierung 243 neue und 181 aus anderen Pflanzen bekannte miRNAs in M. truncatula entdeckt. Zwei dieser miRNAs, miR5229 und miR160f*, sind ausschließlich während der arbuskulären Mykorrhiza zu finden und weitere miRNAs werden während dieser Symbiose verstärkt gebildet. Interessanterweise führen einige dieser miRNAs zum Abbau von Transkripten, die eine wichtige Funktion in der arbuskulären Mykorrhiza und Wurzelknöllchensymbiose besitzen. Die Ergebnisse dieser Studie liefern eine neue Grundlage für die Untersuchung von regulatorischen Netzwerken, die zur zellulären Umprogrammierung während der Interaktion zwischen Pflanzen und arbuskulären Mykorrhiza-Pilzen bei verschiedenen Phosphatbedingungen führen. / AM symbiosis has a positive influence on plant P-nutrition and growth, but little is known about the molecular mechanism of the symbiosis adaptation to different phosphate conditions. The recently described induction of several pri-miR399 transcripts in mycorrhizal shoots and subsequent accumulation of mature miR399 in mycorrhizal roots indicates that local PHO2 expression must be controlled during symbiosis, presumably in order to sustain AM symbiosis development, in spite of locally increased Pi-concentration. A reverse genetic approach used in this study demonstrated that PHO2 and thus the PHR1-miR399-PHO2 signaling pathway, is involved in certain stages of progressive root colonization. In addition, a transcriptomic approach using a split-root system provided a comprehensive insight into the systemic transcriptional changes in mycorrhizal roots and shoots of M. truncatula in response to high phosphate conditions. With regard to the transcriptional responses of the root system, the results indicate that, although the colonization is drastically reduced, AM symbiosis is still functional at high Pi concentrations and might still be beneficial to the plant. Additionally, the data suggest that a specific root-borne mycorrhizal signal systemically induces protein synthesis, amino acid metabolism and photosynthesis at low Pi conditions, which is abolished at high Pi conditions. MiRNAs, such as miR399, are involved in long-distance signaling and are therefore potential systemic signals involved in AM symbiosis. A deep-sequencing approach identified 243 novel miRNAs in the root tissue of M. truncatula. Read-count analysis, qRT-PCR measurements and in situ hybridizations clearly indicated a regulation of miR5229a/b, miR5204, miR160f*, miR160c, miR169 and miR169d*/l*/m*/e.2* during arbuscular mycorrhizal symbiosis. Moreover, miR5204* represses a GRAS TF, which is specifically transcribed in mycorrhizal roots. Since miR5204* is induced by high Pi it might represent a further Pi status-mediating signal beside miR399. This study provides additional evidence that MtNsp2, a key regulator of symbiosis-signaling, is regulated and presumably spatially restricted by miR171h cleavage. In summary, a repression of mycorrhizal root colonization at high phosphate status is most likely due to a repression of the phosphate starvation responses and the loss of beneficial responses in mycorrhizal shoots. These findings provide a new basis for investigating the regulatory network leading to cellular reprogramming during interaction between plants, arbuscular mycorrhizal fungi and different phosphate conditions.

The production of biofuels has increased in recent years, to reduce the dependence on fossil fuels and mitigate climate change. However, current production practices are heavily criticized on their environmental sustainability. Life cycle assessments have therefore been used in policies and academic studies to assess the systems; with divergent results. In the coming years however, biofuel production practices must improve to meet strict environmental sustainability policies. The aims of the research presented in this thesis, are to explore and analyze concepts from industrial symbiosis (IS) to improve the efficiency and environmental performance of biofuel production and identify possible material and energy exchanges between biofuel producers and external industries. An exploration of potential material and energy exchanges resulted in a diverse set of possible exchanges. Many exchanges were identified between biofuel producers to make use of each other’s by-products. There is also large potential for exchanges with external industries, e.g. with the food, energy and chemical producing industries. As such, the biofuel industry and external industries have possibilities for potential collaboration and environmental performance improvements, though implementation of the exchanges may be influenced by many conditions. In order to analyze if concepts from IS can provide benefits to firms of an IS network, an approach was created which outlines how quantifications of IS networks can be produced using life cycle assessment literature for guidelines and methodological considerations. The approach offers methods for quantifying the environmental performance for firms of the IS network and an approach to distribute impacts and credits for the exchanges between firm, to test the assumed benefits for the firms of the IS network. Life cycle assessment, and the approach from this thesis, have been used to quantify the environmental performance of IS networks by building scenarios based on an example from an IS network of biofuel producers in Sweden. From the analyses, it has been found that exchanges of material and energy may offer environmental performance improvements for the IS network and for firms of the network. However, the results are dependent upon the methodological considerations of the assessments, including the reference system, functional unit and allocation methods, in addition to important processes such as the agricultural inputs for the system and energy systems employed. By using industrial symbiosis concepts, biofuel producers have possibilities to improve the environmental performance. This is done by making use of by-products and waste and diversifying their products, promoting a transition toward biorefinery systems and a bio-based economy for regional environmental sustainability. / Produktionen av biobränslen har ökat de senaste åren, vilket är ett steg mot klimateffektivare lösningar i transportsektorn, men biodrivmedlen har ifrågasatts med hänvisning till tveksamheter kring deras miljö- och energiprestanda. Lifecykelanalyser har därför använts inom akademiska studier och för policy för att utvärdera systemen, dock utan samstämmiga resultat. Under de kommande åren måste därför praxis för produktion av biobränslen förbättras för att kunna möta de strikta kraven i hållbarhetskriterier för biobränslen. Syftet med forskningen som presenteras i den här doktorsavhandlingen är att utforska och analysera koncept från området Industriell symbios (IS) och därigenom identifiera förbättringar för ökad effektivitet och miljöprestanda för biobränsleproduktion. Vidare är syftet att identifiera möjliga material- och energiutbyten mellan biobränsleproducenter och externa industrier. Potentiella material- och energiutbyten undersöktes, vilket resulterade förslag på flera olika typer av potentiella utbyten. Undersökningen visar på en potential för att använda biprodukter i en biobränsleprocess som råvara till en annan biobränsleframställning. Vidare identifierades en stor potential för utbyten med externa industrier, som till exempel matproducenter samt industrier för energi och kemikalier. Det är tydligt att det finns möjligheter för biobränsleproducenter och externa industrier att samarbeta och därmed ge möjlighet till förbättringar i miljöprestandan, dock kan en implementering av dessa utbyten påverkas av många olika förutsättningar. Avhandlingen presenterar även ett tillvägagångssätt för att visa hur kvantifiering av miljöprestanda inom ett nätverk för IS kan genomföras genom att använda riktlinjer och metodavvägningar från litteratur för livscykelanalys.  Detta tillvägagångssätt kan användas för att analysera om koncept från IS kan leda till fördelar för företagen i ett IS-nätverk. Tillvägsgångssättet ger möjlighet att kvantifiera miljöprestandan för företagen i IS-nätverket och ger dessutom vägledning för hur miljöpåverkan från utbytena kan distribueras mellan de olika företagen. Metoden utvecklades för att bland annat undersöka de förmodade fördelarna från IS för varje enskild aktör. Livscykelanalys i kombination med tillvägagångssättet ovan har använts för att kvantifiera miljöprestandan för IS-nätverk genom att konstruera scenarier. Scenarierna har baserats på ett exempel från ett IS-nätverk av biobränsleprocenter i Sverige. Analyserna visar att utbyten av material- och energi kan ge förbättringar i miljöprestanda. Resultaten är dock beroende av vilka metodavvägningar som gjorts, till exempel val av referenssystem, funktionell enhet och allokeringsmetoder. Vidare spelar viktiga processer som inputs från jordbruk och val av energisystem stor roll för resultatet. Metodavvägningar för utväderingen influerar även miljöpåverkan samt hur den fördelas mellan företagen i IS-nätverket. Dessutom kan den lokala miljöpåverkan öka medan den globala påverkan minskar. Sammanfattningsvis kan biobränsleproducenter, genom att använda koncept från industriell symbios, ges möjlighet att förbättra sin miljöprestanda. Detta kan ske genom att använda biprodukter och avfall samt genom att diversifiera sina produkter som ett första steg mot en övergång mot bioraffinaderier och en mer biobaserad ekonomi för regional hållbarhet.

The colonial marine bryozoan, Bugula neritina, harbors an uncultured endosymbiont, “Candidatus Endobugula sertula” throughout its life stages. The bacterial symbiont has been proposed to be a source of complex polyketide metabolites, the bryostatins, that chemically defend B. neritina larvae from predation. Within a bryozoan colony, significantly higher amounts of bryostatins are found in ovicell-bearing zooids where the developing larvae are brooded, as compared to ovicell-free zooids. It is hypothesized that signaling between B. neritina and “Ca. Endobugula sertula” may be involved in the regulation of bryostatin production in different zooids, as well as in maintenance of the symbiosis. In this study, suppression subtractive hybridization (SSH) was used to identify differentially expressed host genes during this association. The identified genes suggest that the host plays a role in the distribution and localization of bacterial symbionts in different host zooids, possibly to regulate levels of bryostatin production in the zooids.

Root symbioses with dark septate endophytic fungi (DSE) and arbuscular mycorrhizal fungi (AMF) provide plant tolerance to environmental stresses. This research answers several fundamental questions about the occurrence of these fungi in roots of prairie grasses. Traditional methods and current molecular techniques were combined in order to: 1) define the role and specificity of DSE in plant tolerance to drought; 2) assess the level of host specificity in DSE; 3) document AMF biodiversity and pattern of root colonization at different soil depths; 4) define the influence of soil depth and plant species on the distribution of DSE and AMF in roots and; 5) reveal how DSE and AMF interact in plant roots.<p> Under controlled conditions, DSE isolates showed host preference in colonizing roots and promoting plant growth. They colonized with more intensity the plant species from which they were isolated [Agropyron cristatum L. or Psathyrostachys juncea (Fisch) Nevski subsp. Juncea (Syn: Elymus junceus Fisch)]. Inoculation with five DSE isolates resulted in growth stimulation of the C3 grasses A. cristatum and P. juncea, and growth depression of the C4 grass Bouteloua gracillis (Willd. ex Kunth) Lag. ex Griffiths, under water stress. Plant C concentration suggested that DSE inoculation may have resulted in net C drain from B. gracillis.<p. In the field, soil depth influenced root colonization in A. cristatum, Panicum virgatum L., Nassella viridula Trin and Pascopyrum smithii (Rydb.) A. Löve., while AMF diversity was influenced by the interaction between soil depth and host plant species. Molecular analysis of roots serially sampled during one growing season from the A and B soil horizons, in stands of these grasses, revealed spatial and temporal changes in DSE and AMF community composition, and a significant correlation in DSE and AMF community structure.<p> These results suggest that DSE and AMF are adapted to specific environmental conditions and that root occupation by these fungi is a dynamic phenomenon. It is proposed that temporal variation in root occupation by DSE and AMF impacts plant and ecosystem processes at different times during the growing season.

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

Root symbioses with dark septate endophytic fungi (DSE) and arbuscular mycorrhizal fungi (AMF) provide plant tolerance to environmental stresses. This research answers several fundamental questions about the occurrence of these fungi in roots of prairie grasses. Traditional methods and current molecular techniques were combined in order to: 1) define the role and specificity of DSE in plant tolerance to drought; 2) assess the level of host specificity in DSE; 3) document AMF biodiversity and pattern of root colonization at different soil depths; 4) define the influence of soil depth and plant species on the distribution of DSE and AMF in roots and; 5) reveal how DSE and AMF interact in plant roots.<p> Under controlled conditions, DSE isolates showed host preference in colonizing roots and promoting plant growth. They colonized with more intensity the plant species from which they were isolated [Agropyron cristatum L. or Psathyrostachys juncea (Fisch) Nevski subsp. Juncea (Syn: Elymus junceus Fisch)]. Inoculation with five DSE isolates resulted in growth stimulation of the C3 grasses A. cristatum and P. juncea, and growth depression of the C4 grass Bouteloua gracillis (Willd. ex Kunth) Lag. ex Griffiths, under water stress. Plant C concentration suggested that DSE inoculation may have resulted in net C drain from B. gracillis.<p. In the field, soil depth influenced root colonization in A. cristatum, Panicum virgatum L., Nassella viridula Trin and Pascopyrum smithii (Rydb.) A. Löve., while AMF diversity was influenced by the interaction between soil depth and host plant species. Molecular analysis of roots serially sampled during one growing season from the A and B soil horizons, in stands of these grasses, revealed spatial and temporal changes in DSE and AMF community composition, and a significant correlation in DSE and AMF community structure.<p> These results suggest that DSE and AMF are adapted to specific environmental conditions and that root occupation by these fungi is a dynamic phenomenon. It is proposed that temporal variation in root occupation by DSE and AMF impacts plant and ecosystem processes at different times during the growing season.

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

The steps by which neutral, random and/or negative biological interactions evolve into mutualistic ones remain poorly understood. Here, we study Elacatinus gobies and the fishes from which they clean parasites, termed 'clients'. Colorful stripes are common to mutualist cleaners and non-cleaning sister species. Blue stripes are unique to obligate cleaners. We quantified the contrast potential of ancestral and novel stripe colors, using fish color vision models, and determined that color stripes have become more visible to clients over evolutionary time. In turn, we focused on the role of color as a potentially specialized signal. We show that cleaners possess a putative chemical defense (one multimedia file in .mov format included) and demonstrate that stripes are sufficient to elicit client stereotypical posing behavior and to deter attack. Analysis of previously published records show that yellow cleaners tend to predators less than expected, compared to green and blue cleaners. Our results highlight evolution from predator avoidance to tolerance with conspicuous advertising reinforced by chemical defense in cleaners. Similar trajectories, via recognizable signals to risky partners, may be common in other diffuse mutualisms. We discuss the generality of defense and signal traits in other species rich lineages of mutualists.