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Beneficial soil-borne microorganisms, such as plant growth promoting rhizobacteria and mycorrhizal fungi, can improve plant performance by inducing systemic defense responses that confer broad-spectrum resistance to plant pathogens and even insect herbivores. Different beneficial microbe-associated molecular patterns (MAMPs) are recognized by the plant, which results in a mild, but effective activation of the plant immune responses in systemic tissues. Evidence is accumulating that systemic resistance induced by different beneficials is regulated by similar jasmonate-dependent and ethylene-dependent signaling pathways and is associated with priming for enhanced defense.

Rhizobacteria–induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

Cross talk between salicylic acid (SA) and jasmonic acid (JA) signaling pathways plays an important role in the regulation and fine tuning of induced defenses that are activated upon pathogen or insect attack. Pharmacological experiments revealed that transcription of JA-responsive marker genes, such as PDF1.2 and VSP2, is highly sensitive to suppression by SA. This antagonistic effect of SA on JA signaling was also observed when the JA pathway was biologically activated by necrotrophic pathogens or insect herbivores, and when the SA pathway was triggered by a biotrophic pathogen. Furthermore, all 18 Arabidopsis (Arabidopsis thaliana) accessions tested displayed SA-mediated suppression of JA-responsive gene expression, highlighting the potential significance of this phenomenon in induced plant defenses in nature. During plant-attacker interactions, the kinetics of SA and JA signaling are highly dynamic. Mimicking this dynamic response by applying SA and methyl jasmonate (MeJA) at different concentrations and time intervals revealed that PDF1.2 transcription is readily suppressed when the SA response was activated at or after the onset of the JA response, and that this SA-JA antagonism is long lasting. However, when SA was applied more than 30 h prior to the onset of the JA response, the suppressive effect of SA was completely absent. The window of opportunity of SA to suppress MeJA-induced PDF1.2 transcription coincided with a transient increase in glutathione levels. The glutathione biosynthesis inhibitor l-buthionine-sulfoximine strongly reduced PDF1.2 suppression by SA, suggesting that SA-mediated redox modulation plays an important role in the SA-mediated attenuation of the JA signaling pathway.

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture. © 2006 Elsevier Inc. All rights reserved.

SummaryThe maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.

The present publication deals with recent palynological results of a relatively small interval of Muschelkalk and exposures of the overlying clay deposits in the Winterswijk quarries. For the first time the Lower Muschelkalk Member in the Netherlands could be independently dated as Bithynian (Anisian, Middle Triassic). Contrary to widely accepted opinions the overlying almost black clay deposit is not Liassic but Rhaetian in age and it is assigned to the Sleen Shale Formation. This marginal marine clay which pinches out to the south, is in turn overlain by a light gray, full-marine Lower-Oligocene clay of the Rupel Formation. An anomalous occurrence of Liassic clay is now attributed to subrosion of Röt salt followed by collapse of the overlying Muschelkalk, Rhaetian and Lias strata.

Elevated CO2 enhances photosynthesis and growth of plants, but the enhancement is strongly influenced by the availability of nitrogen. In this article, we summarise our studies on plant responses to elevated CO2. The photosynthetic capacity of leaves depends not only on leaf nitrogen content but also on nitrogen partitioning within a leaf. In Polygonum cuspidatum, nitrogen partitioning among the photosynthetic components was not influenced by elevated CO2 but changed between seasons. Since the alteration in nitrogen partitioning resulted in different CO2-dependence of photosynthetic rates, enhancement of photosynthesis by elevated CO2 was greater in autumn than in summer. Leaf mass per unit area (LMA) increases in plants grown at elevated CO2. This increase was considered to have resulted from the accumulation of carbohydrates not used for plant growth. With a sensitive analysis of a growth model, however, we suggested that the increase in LMA is advantageous for growth at elevated CO2 by compensating for the reduction in leaf nitrogen concentration per unit mass. Enhancement of reproductive yield by elevated CO2 is often smaller than that expected from vegetative growth. In Xanthium canadense, elevated CO2 did not increase seed production, though the vegetative growth increased by 53%. As nitrogen concentration of seeds remained constant at different CO 2 levels, we suggest that the availability of nitrogen limited seed production at elevated CO2 levels. We found that leaf area development of plant canopy was strongly constrained by the availability of nitrogen rather than by CO2. In a rice field cultivated at free-air CO2 enrichment, the leaf area index (LAI) increased with an increase in nitrogen availability but did not change with CO2 elevation. We determined optimal LAI to maximise canopy photosynthesis and demonstrated that enhancement of canopy photosynthesis by elevated CO2 was larger at high than at low nitrogen availability. We also studied competitive asymmetry among individuals in an even-aged, monospecific stand at elevated CO 2. Light acquisition (acquired light per unit aboveground mass) and utilisation (photosynthesis per unit acquired light) were calculated for each individual in the stand. Elevated CO2 enhanced photosynthesis and growth of tall dominants, which reduced the light availability for shorter subordinates and consequently increased size inequality in the stand.

Summary The need for evolutionary studies on quantitative traits that integrate genetics is increasing. Studies on consistent individual differences in behavioural traits provide a good opportunity to do controlled experiments on the genetic mechanisms underlying the variation and covariation in complex behavioural traits. In this review we will highlight the contribution of genetic studies in animal personality research. We will start with reviewing the evidence that shows how much variation in animal personality traits is genetic, and connect this to knowledge from human personality studies. We will continue by considering the nature of that variation, its generation and maintenance. Finally we will point to further possibilities for studying the genetics of animal personalities. We will underline the importance of integrating both proximate and ultimate approaches when studying the evolution of animal personalities.

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.

We analyzed fossil chironomids (nonbiting midges) and pollen in two lake-sediment records to reconstruct and quantify Holocene summer-temperature fluctuations in the European Alps. Chironomid and pollen records indicate five centennial-scale cooling episodes during the early- and mid-Holocene. The strongest temperature declines of ≈1°C are inferred at ≈10,700–10,500 and 8,200–7,600 calibrated 14 C years B.P., whereas other temperature fluctuations are of smaller amplitude. Two forcing mechanisms have been presented recently to explain centennial-scale climate variability in Europe during the early- and mid-Holocene, both involving changes in Atlantic thermohaline circulation. In the first mechanism, changes in meltwater flux from the North American continent to the North Atlantic are responsible for changes in the Atlantic thermohaline circulation, thereby affecting circum-Atlantic climate. In the second mechanism, solar variability is the cause of Holocene climatic fluctuations, possibly triggering changes in Atlantic thermohaline overturning. Within their dating uncertainty, the two major cooling periods in the European Alps are coeval with substantial changes in the routing of North American freshwater runoff to the North Atlantic, whereas quantitatively, our climatic reconstructions show a poor agreement with available records of past solar activity. Thus, our results suggest that, during the early- and mid-Holocene, freshwater-induced Atlantic circulation changes had stronger influence on Alpine summer temperatures than solar variability and that Holocene thermohaline circulation reductions have led to summer-temperature declines of up to 1°C in central Europe.

Within their environment, plants interact with a wide range of microorganisms, some of which are pathogenic and cause disease, and others that are beneficial and stimulate plant growth or activate natural defenses. To recognize and respond to this variety of pathogenic and beneficial microorganisms, plants have developed sophisticated strategies to “perceive” microorganisms and translate that “perception” into an appropriate adaptive response. This plant innate immune response is surprisingly complex and highly flexible in its capacity to recognize and respond to different invaders. Jasmonic acid and derivatives, collectively called jasmonates (JAs), have emerged as important signals in the regulation of plant responses to pathogenic and beneficial microorganisms. The complex interplay of JAs with the alarm signals salicylic acid (SA) and ethylene (ET) provides plants with a regulatory potential that shapes the ultimate outcome of the plant-microbe interaction. In this review, we present an overview of the key role of JAs in basal and induced resistance to pathogens, their possible implication in the establishment and functioning of beneficial plant-microbe associations; and our current knowledge on how the JA signaling pathway cross-communicates with SA- and ET-dependent signaling pathways to fine-tune defense.

Within the framework of a palaeolimnological pilot study, the potential of sedimentary diatom assemblages for the reconstruction of the eutrophication history was studied in short sediment cores from five shallow lakes located in the Romanian Danube Delta. A total of 234 diatom taxa representing 57 genera could be identified but diatom assemblages are generally predominated by a few species only. Loss on ignition percentages, total diatom abundances and diatom downcore successions largely vary within the single sediment cores and between all five lakes. This suggests the presence of various lake environments within the Danube Delta, each characterized by lake-specific geomorphological, sedimentological, hydrological, and biological conditions. Highly variable depositional conditions are also confirmed by radiocesium measurements in two of the five cores suggesting distinct inter-lake differences in the sedimentation rate. Causes for the great intra-lake and inter-lake variability in diatom downcore successions are difficult to specify and may include changes in the physico-chemical conditions, aquatic vegetation cover, water depth, river influence, turbidity and inter-biotic interactions. However, based on this pilot study, it is likely that the spatial and temporal distribution of sedimentary diatom assemblages in Danube Delta lakes were not triggered by the trophic state. The delta lakes likely became meso- to eutrophic long before 1950, possibly during late-Holocene times.

Contains fulltext : 60109.pdf (Publisher’s version ) (Open Access) Plants growing at high densities express shade avoidance traits as a response to the presence of neighbours. Enhanced shoot elongation is one of the best researched shade avoidance components and increases light capture in dense stands. We show here that also leaf movements, leading to a more vertical leaf orientation (hyponasty), may be crucial in the early phase of competition. The initiation of shade avoidance responses is classically attributed to the action of phytochrome photoreceptors that sense red:far-red (R:FR) ratios in light reflected by neighbours, but also other signals may be involved. It was recently shown that ethylene-insensitive, transgenic (Tetr) tobacco plants, which are insensitive to the gaseous plant hormone ethylene, have reduced shade avoidance responses to neighbours. Here, we report that this is not related to a reduced response to low R:FR ratio, but that Tetr tobacco plants are unresponsive to a reduced photon fluence rate of blue light, which normally suppresses growth inhibition in wild-type (WT) plants. In addition to these light signals, ethylene levels in the canopy atmosphere increased to concentrations that could induce shade avoidance responses in WT plants. Together, these data show that neighbour detection signals other than the R:FR ratio are more important than previously anticipated and argue for a particularly important role for ethylene in determining plant responses to neighbours.

The northwest Iberian ocean margin experiences seasonal upwelling of nutrient-rich water resulting in enhanced primary productivity. The quantitative organic-walled dinoflagellate cyst distribution and that of other palynomorphs has been determined in 37 box core-tops along three transects to trace the upwelling signal. Oligotrophic Impagidinium spp. represent the dominant offshore elements. Cysts of heterotrophic Protoperidinium spp. mainly occur in sediments of the seasonal upwelling area on the shelf and slope and show a sharp decrease offshore. The eutrophic autotrophic taxon Lingulodinium machaerophorum is also abundant in sediments of the seasonal upwelling area on the shelf and slope, and also decreases more offshore. Both relative as well as absolute numbers of these taxa may be used to trace the upwelling system despite potential taphonomic problems. Considering the overall similar environmental conditions with respect to the other two transects, the elevated palynomorph concentrations in the shielded Nazare Canyon samples may be seen to represent the actual fluxes of palynomorphs along the Iberian coast. If accepted, this would indicate a preservational and transport loss of cysts of an order of magnitude in other transects.

Water and nutrients required by developing seeds are mainly supplied by the phloem and have to be released from a maternal parenchyma tissue before being utilized by the filial tissues of embryo and endosperm. To identify aquaporins that could be involved in this process four full-length cDNAs were cloned and sequenced from a cDNA library of developing seed coats of pea (Pisum sativum L.). The cDNA of PsPIP1-1 appeared to be identical to that of clone 7a/TRG-31, a turgor-responsive gene cloned previously from pea roots. PsPIP1-1, PsPIP2-1, and PsTIP1-1, or their possible close homologues, were also expressed in cotyledons of developing and germinating seeds, and in roots and shoots of seedlings, but transcripts of PsNIP-1 were only detected in the seed coat. In mature dry seeds, high hybridization signals were observed with the probe for PsPIP1-1, but transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1 were not detected. Functional characterization after heterologous expression in Xenopus oocytes showed that PsPIP2-1 and PsTIP1-1 are aquaporins whereas PsNIP-1 is an aquaglyceroporin. PsNIP-1, like several other NIPs, contains a tryptophan residue corresponding with Trp-48 in GlpF (the glycerol facilitator of Escherichia coli) that borders the selectivity filter in the permeation channel. It is suggested that PsPIP1-1 and/or its possible close homologues could play a role in water absorption during seed imbibition, and that PsPIP2-1, possibly together with PsPIP1-1, could be involved in the release of phloem water from the seed coat symplast, which is intimately connected with the release of nutrients for the embryo. Abbreviations: MIPs, major intrinsic proteins; NIPs, nodulin 26-like intrinsic proteins; PIPs, plasma membrane intrinsic proteins; SIPs, small, basic intrinsic proteins; TIPs, tonoplast intrinsic proteins

Specific strains of nonpathogenic rhizobacteria can induce systemic resistance that is effective against a range of plant pathogens. To exploit induced systemic resistance, detailed knowledge of the triggering bacterial traits involved and on signal transduction pathways in the plant is necessary. Possibilities to improve effectiveness of induced resistance by rhizobacterial strains are discussed.

We report an experimental and microstructural study of the frictional properties of simulated fault gouges prepared from natural limestone (96 % CaCO3) and pure calcite. Our experiments consisted of direct shear tests performed, under dry and wet conditions, at an effective normal stress of 50 MPa, at 18–150 °C and sliding velocities of 0.1–10 μm/s. Wet experiments used a pore water pressure of 10 MPa. Wet gouges typically showed a lower steady-state frictional strength (μ = 0.6) than dry gouges (μ = 0.7–0.8), particularly in the case of the pure calcite samples. All runs showed a transition from stable velocity strengthening to (potentially) unstable velocity weakening slip above 80–100 °C. All recovered samples showed patchy, mirror-like surfaces marking boundary shear planes. Optical study of sections cut normal to the shear plane and parallel to the shear direction showed both boundary and inclined shear bands, characterized by extreme grain comminution and a crystallographic preferred orientation. Cross-sections of boundary shears, cut normal to the shear direction using focused ion beam—SEM, from pure calcite gouges sheared at 18 and 150 °C, revealed dense arrays of rounded, ~0.3 μm-sized particles in the shear band core. Transmission electron microscopy showed that these particles consist of 5–20 nm sized calcite nanocrystals. All samples showed evidence for cataclasis and crystal plasticity. Comparing our results with previous models for gouge friction, we suggest that frictional behaviour was controlled by competition between crystal plastic and granular flow processes active in the shear bands, with water facilitating pressure solution, subcritical cracking and intergranular lubrication. Our data have important implications for the depth of the seismogenic zone in tectonically active limestone terrains. Contrary to recent claims, our data also demonstrate that nanocrystalline mirror-like slip surfaces in calcite(-rich) faults are not necessarily indicative of seismic slip rates.

We report an experimental and microstructural study of the frictional properties of simulated fault gouges prepared from natural limestone (96 % CaCO3) and pure calcite. Our experiments consisted of direct shear tests performed, under dry and wet conditions, at an effective normal stress of 50 MPa, at 18–150 °C and sliding velocities of 0.1–10 μm/s. Wet experiments used a pore water pressure of 10 MPa. Wet gouges typically showed a lower steady-state frictional strength (μ = 0.6) than dry gouges (μ = 0.7–0.8), particularly in the case of the pure calcite samples. All runs showed a transition from stable velocity strengthening to (potentially) unstable velocity weakening slip above 80–100 °C. All recovered samples showed patchy, mirror-like surfaces marking boundary shear planes. Optical study of sections cut normal to the shear plane and parallel to the shear direction showed both boundary and inclined shear bands, characterized by extreme grain comminution and a crystallographic preferred orientation. Cross-sections of boundary shears, cut normal to the shear direction using focused ion beam—SEM, from pure calcite gouges sheared at 18 and 150 °C, revealed dense arrays of rounded, ~0.3 μm-sized particles in the shear band core. Transmission electron microscopy showed that these particles consist of 5–20 nm sized calcite nanocrystals. All samples showed evidence for cataclasis and crystal plasticity. Comparing our results with previous models for gouge friction, we suggest that frictional behaviour was controlled by competition between crystal plastic and granular flow processes active in the shear bands, with water facilitating pressure solution, subcritical cracking and intergranular lubrication. Our data have important implications for the depth of the seismogenic zone in tectonically active limestone terrains. Contrary to recent claims, our data also demonstrate that nanocrystalline mirror-like slip surfaces in calcite(-rich) faults are not necessarily indicative of seismic slip rates.

Selected nonpathogenic rhizobacteria with biological disease control activity are able to elicit an induced systemic resistance (ISR) response that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Ten ecotypes of Arabidopsis thaliana were screened for their potential to express rhizobacteria-mediated ISR and pathogen-induced SAR against the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). All ecotypes expressed SAR. However, of the 10 ecotypes tested, ecotypes RLD and Wassilewskija (Ws) did not develop ISR after treatment of the roots with nonpathogenic Pseudomonas fluorescens WCS417r bacteria. This nonresponsive phenotype was associated with relatively high susceptibility to Pst infection. The F1 progeny of crosses between the non-responsive ecotypes RLD and Ws on the one hand, and the responsive ecotypes Columbia (Col) and Landsberg erecta (Ler) on the other hand, were fully capable of expressing ISR and exhibited a relatively high level of basal resistance, similar to that of their WCS417r-responsive parent. This indicates that the potential to express ISR and the relatively high level of basal resistance against Pst are both inherited as dominant traits. Analysis of the F2 and F3 progeny of a Col × RLD cross revealed that inducibility of ISR and relatively high basal resistance against Pst cosegregate in a 3 : 1 fashion, suggesting that both resistance mechanisms are monogenically determined and genetically linked. Neither the responsiveness to WCS417r nor the relatively high level of basal resistance against Pst were complemented in the F1 progeny of crosses between RLD and Ws, indicating that RLD and Ws are both affected in the same locus, necessary for the expression of ISR and basal resistance against Pst. The corresponding locus, designated ISR1, was mapped between markers B4 and GL1 on chromosome 3. The observed association between ISR and basal resistance against Pst suggests that rhizo-bacteria-mediated ISR against Pst in Arabidopsis requires the presence of a single dominant gene that functions in the basal resistance response against Pst infection.

Stearoyl-CoA desaturase (SCD) in the bovine mammary gland introduces a cis-double bond at the ¿9 position in a wide range of fatty acids (FA). Several long-chain polyunsaturated fatty acids (PUFA) inhibit expression of SCD, but information on the effect of short-chain fatty acids on mammary SCD expression is scarce. We used a bovine mammary cell line (MAC-T) to assess the effect of acetic acid (Ac) and ß-hydroxybutyric acid (BHBA) in comparison with the effect of various long-chain fatty acids on the mRNA expression of the lipogenic enzymes SCD, acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and their associated gene regulatory proteins sterol regulatory element binding transcription factor 1 (SREBF1), insulin-induced gene 1 protein (INSIG1) and peroxisome proliferator-activated receptor alpha (PPARA)and peroxisome proliferator-activated receptor delta (PPARD) by quantitative real-time PCR. MAC-T cells were treated for 12 h without FA additions (CON) or with either 5 mM Ac, 5 mM BHBA, a combination of 5 mM Ac + 5 mM BHBA, 100 µM C16:0, 100 µM C18:0, 100 µM C18:1 cis-9, 100 µM C18:1 trans-11, 100 µM C18:2 cis-9,12 or 100 µM C18:3 cis-9,12,15. Compared with control, mRNA expression of SCD1 was increased by Ac (+61%) and reduced by C18:1 cis-9 (-61%), C18:2 cis-9,12 (-84%) and C18:3 cis-9,12,15 (-88%). In contrast to native bovine mammary gland tissue, MAC-T cells did not express SCD5. Expression of ACACA was increased by Ac (+44%) and reduced by C18:2 cis-9,12 (-48%) and C18:3 cis-9,12,15 (-49%). Compared with control, FASN expression was not significantly affected by the treatments. The mRNA level of SREBF1 was not affected by Ac or BHBA, but was reduced by C18:1 cis-9 (-44%), C18:1 trans-11 (-42%), C18:2 cis-9,12 (-62%) and C18:3 cis-9,12,15 (-68%) compared with control. Expression of INSIG1 was downregulated by C18:0 (-37%), C18:1 cis-9 (-63%), C18:1 trans-11 (-53%), C18:2 cis-9,12 (-81%) and C18:3 cis-9,12,15 (-91%). Both PPARA and PPARD expression were not significantly affected by the treatments. Our results show that Ac upregulated mRNA expression of SCD1 and ACACA in MAC-T cells. The opposite effect of the PUFA C18:2 cis-9,12 and C18:3 cis-9,12,15 on the these genes and the failure of Ac to mimic the PUFA-inhibited SREBF1 and INSIG1 mRNA expression, suggest that Ac can stimulate mammary lipogenesis via a transcriptional regulatory mechanism different from PUFA.

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis-double bond at the ¿9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non-invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein-Friesian cows and used for analysis of SCD expression by real-time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to ¿9-desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non-invasive alternative to mammary tissue samples obtained by biopsy.

A comparative non-ruminant species view of the contribution of the large intestinal metabolism to inaccuracies in nitrogen and amino acid absorption measurements is provided to assess potential implications for the determination of crude protein/amino acid digestibility in adult humans consuming lower digestible protein sources. Most of the amino acids in the hindgut are constituents of the microorganisms and significant microbial metabolism of dietary and endogenous amino acids occurs. Bacterial metabolism of nitrogen-containing compounds leads to a significant disappearance of nitrogen in the large intestine. Literature data show that some 79 % of the nitrogen entering the large intestine of the horse is absorbed. For dogs, sows, and growing pigs these estimates are 49, 34 and 16 %, respectively. The coefficient of gut differentiation of humans compares closely to that of dogs while the coefficient of fermentation in humans is the lowest of all non-ruminant species and closest to that of cats and dogs. Large intestinal digesta transit times of humans compare closest to adult dogs. Significant amino acid metabolism has been shown to occur in the large intestine of the adult dog. Use of the growing pig as an animal model is likely to underestimate the fermentation of amino acids in the human large intestine. Based on the significant degree of fermentation of nitrogen-containing components in the large intestine of several non-ruminant species, it can be expected that determination of amino acid digestibility at a faecal level in humans consuming low quality proteins would not provide accurate estimates of the amino acids absorbed by the intestine.

The vertebrate immune system uses several strategies to combat viral infections. Cellular immune responses evoked by the cytotoxic T cells (CTLs) are initiated after screening host cells for abnormalities. Examples are peptides derived from virus (epitopes) that are presented on the cell surface by major histocompability molecules (MHCs). Viruses therefore face strong selection pressures to evade the pathways signaling such abnormalities to the immune cells. In this thesis we address the co-evolutionof pathogens and hosts of one such a pathway, i.e., the classical Ag presentation pathway. The recent adaptation of HIV-1 to its new human host was studied by enumerating the total number of predicted CTL epitopes and epitope precursors in a HIV-1 sequences gathered over more than 25 years. We investigated the adaptation of HIV-1 by comparing the distribution of CTL epitopes in HIV-1 proteins with other proteins, and found no conclusive evidence for any ongoing accumulation of CTL epitope escape mutations in HIV-1. This was surprising because two important molecules in the pathway (proteasome and TAP) display hardly any genetic variation between individuals, and a rapidly evolving virus like HIV-1 should in 25 years be able to evolve an escape from such monomorphic molecules. We hypothesized that the virus fails to escape from the monomorphic steps of the pathway because the most specific molecule in this pathway, i.e., the MHC, is highly polymorphic (i.e., is genetically diverse in the human population). Upon transmission of virus to a new host, previous proteasome and TAP escape mutations are released from immune selection pressure because the new host's MHC selects another set peptides from HIV-1's proteome than the previous host. This scenario was tested in an individual-based computer simulation model, in which an HIV-1 like virus was allowed to adapt to a human like host population. We showed that different degrees of MHC polymorphism in the host population affected how much the virus was adapting to the polymorphic MHC, or to the monomorphic proteasome and TAP. Finally, we extended the model such that the host population could evolve all components of its antigen presentation pathway in response to multiple endemic pathogens. This allowed us to study whether the current structure of the human antigen presentation pathway could be explained just in terms of host-pathogen co-evolution. Based on our earlier hypothesized mechanism, we expected that only the most specific step of the pathway would evolve a polymorphism. Under the constraint that the individual steps of the pathway remain co-evolved, it was indeed only the most specific step of the Ag presentation pathway that became polymorphic.

The vertebrate immune system uses several strategies to combat viral infections. Cellular immune responses evoked by the cytotoxic T cells (CTLs) are initiated after screening host cells for abnormalities. Examples are peptides derived from virus (epitopes) that are presented on the cell surface by major histocompability molecules (MHCs). Viruses therefore face strong selection pressures to evade the pathways signaling such abnormalities to the immune cells. In this thesis we address the co-evolutionof pathogens and hosts of one such a pathway, i.e., the classical Ag presentation pathway. The recent adaptation of HIV-1 to its new human host was studied by enumerating the total number of predicted CTL epitopes and epitope precursors in a HIV-1 sequences gathered over more than 25 years. We investigated the adaptation of HIV-1 by comparing the distribution of CTL epitopes in HIV-1 proteins with other proteins, and found no conclusive evidence for any ongoing accumulation of CTL epitope escape mutations in HIV-1. This was surprising because two important molecules in the pathway (proteasome and TAP) display hardly any genetic variation between individuals, and a rapidly evolving virus like HIV-1 should in 25 years be able to evolve an escape from such monomorphic molecules. We hypothesized that the virus fails to escape from the monomorphic steps of the pathway because the most specific molecule in this pathway, i.e., the MHC, is highly polymorphic (i.e., is genetically diverse in the human population). Upon transmission of virus to a new host, previous proteasome and TAP escape mutations are released from immune selection pressure because the new host's MHC selects another set peptides from HIV-1's proteome than the previous host. This scenario was tested in an individual-based computer simulation model, in which an HIV-1 like virus was allowed to adapt to a human like host population. We showed that different degrees of MHC polymorphism in the host population affected how much the virus was adapting to the polymorphic MHC, or to the monomorphic proteasome and TAP. Finally, we extended the model such that the host population could evolve all components of its antigen presentation pathway in response to multiple endemic pathogens. This allowed us to study whether the current structure of the human antigen presentation pathway could be explained just in terms of host-pathogen co-evolution. Based on our earlier hypothesized mechanism, we expected that only the most specific step of the pathway would evolve a polymorphism. Under the constraint that the individual steps of the pathway remain co-evolved, it was indeed only the most specific step of the Ag presentation pathway that became polymorphic.

The vertebrate immune system uses several strategies to combat viral infections. Cellular immune responses evoked by the cytotoxic T cells (CTLs) are initiated after screening host cells for abnormalities. Examples are peptides derived from virus (epitopes) that are presented on the cell surface by major histocompability molecules (MHCs). Viruses therefore face strong selection pressures to evade the pathways signaling such abnormalities to the immune cells. In this thesis we address the co-evolutionof pathogens and hosts of one such a pathway, i.e., the classical Ag presentation pathway. The recent adaptation of HIV-1 to its new human host was studied by enumerating the total number of predicted CTL epitopes and epitope precursors in a HIV-1 sequences gathered over more than 25 years. We investigated the adaptation of HIV-1 by comparing the distribution of CTL epitopes in HIV-1 proteins with other proteins, and found no conclusive evidence for any ongoing accumulation of CTL epitope escape mutations in HIV-1. This was surprising because two important molecules in the pathway (proteasome and TAP) display hardly any genetic variation between individuals, and a rapidly evolving virus like HIV-1 should in 25 years be able to evolve an escape from such monomorphic molecules. We hypothesized that the virus fails to escape from the monomorphic steps of the pathway because the most specific molecule in this pathway, i.e., the MHC, is highly polymorphic (i.e., is genetically diverse in the human population). Upon transmission of virus to a new host, previous proteasome and TAP escape mutations are released from immune selection pressure because the new host's MHC selects another set peptides from HIV-1's proteome than the previous host. This scenario was tested in an individual-based computer simulation model, in which an HIV-1 like virus was allowed to adapt to a human like host population. We showed that different degrees of MHC polymorphism in the host population affected how much the virus was adapting to the polymorphic MHC, or to the monomorphic proteasome and TAP. Finally, we extended the model such that the host population could evolve all components of its antigen presentation pathway in response to multiple endemic pathogens. This allowed us to study whether the current structure of the human antigen presentation pathway could be explained just in terms of host-pathogen co-evolution. Based on our earlier hypothesized mechanism, we expected that only the most specific step of the pathway would evolve a polymorphism. Under the constraint that the individual steps of the pathway remain co-evolved, it was indeed only the most specific step of the Ag presentation pathway that became polymorphic.

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase.

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase.

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase.

Puberty is the process by which a juvenile acquires for the first time the capacity to reproduce sexually, a critical basis for the conservation of a species. In general, the biological processes constituting the first gonadal maturation and the mechanisms involved in this process are similar among vertebrates. Internal and/or external signals trigger the secretion of gonadotropin-releasing hormone (Gnrh) by neurosecretory neurons in the ventral forebrain, and Gnrh stimulates the synthesis and secretion of two pituitary hormones, the gonadotropins known as follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). These two glycoproteins are the major hormones regulating the remarkable structural and functional changes occurring in the pubertal gonads during the activation of the two principal gonad tasks, the production of gametes and sex steroids. In context with the male-biased problems of precocious testis maturation in farmed cod, the present thesis focuses on male puberty. Pituitary fsh gene expression starts to increase at the beginning of testis development (proliferation of spermatogonia), followed by a slow elevation in testosterone plasma levels. With rapidly continuing spermatogenesis, which takes place in a peculiar spatio-temporal organization in the testicular lobes, high gonado-somatic indexes of up to 16 % are attained. Close to spawning, testosterone and 11-ketotestosterone, two potent ligands for the cod androgen receptor (Ar), peak in parallel with pituitary lh and testicular lhr (Lh receptor) gene expression, while fshr seems to be expressed in a constitutively manner in the cod testis. It is possible that attaining high plasma levels of potent androgens involves an auto-protective manner of androgen production, where weak Ar ligands, such as androstenedione and 11-ketoandrostenedione, are produced by the testis, thereby avoiding androgen-induced down-regulation of steroidogenic enzymes, while conversion to 17߭hydroxylated, potent androgens would occur in extra-gonadal tissues. After spawning, pituitary gonadotropin expression and androgens plasma levels decrease to basal levels, and only non-proliferating type A spermatogonia are present as germ cell population in the testis, conditions characterizing the spent condition. Like in many other teleost fish living at high latitudes, seasonally changing parameters are crucial environmental cues for cod. In this context, the present thesis also provides important information on the effects of constant light, a commonly used tool to prevent gonad maturation in cod farms, on the activity of the pituitary-testis axis. For instance, we found that even under constant light conditions, some males can escape the light-mediated inhibition and develop, associated with elevated pituitary lh and testicular ar expressions, spermatogenic activity including sperm production, albeit in a qualitatively and quantitatively compromised manner.

Puberty is the process by which a juvenile acquires for the first time the capacity to reproduce sexually, a critical basis for the conservation of a species. In general, the biological processes constituting the first gonadal maturation and the mechanisms involved in this process are similar among vertebrates. Internal and/or external signals trigger the secretion of gonadotropin-releasing hormone (Gnrh) by neurosecretory neurons in the ventral forebrain, and Gnrh stimulates the synthesis and secretion of two pituitary hormones, the gonadotropins known as follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). These two glycoproteins are the major hormones regulating the remarkable structural and functional changes occurring in the pubertal gonads during the activation of the two principal gonad tasks, the production of gametes and sex steroids. In context with the male-biased problems of precocious testis maturation in farmed cod, the present thesis focuses on male puberty. Pituitary fsh gene expression starts to increase at the beginning of testis development (proliferation of spermatogonia), followed by a slow elevation in testosterone plasma levels. With rapidly continuing spermatogenesis, which takes place in a peculiar spatio-temporal organization in the testicular lobes, high gonado-somatic indexes of up to 16 % are attained. Close to spawning, testosterone and 11-ketotestosterone, two potent ligands for the cod androgen receptor (Ar), peak in parallel with pituitary lh and testicular lhr (Lh receptor) gene expression, while fshr seems to be expressed in a constitutively manner in the cod testis. It is possible that attaining high plasma levels of potent androgens involves an auto-protective manner of androgen production, where weak Ar ligands, such as androstenedione and 11-ketoandrostenedione, are produced by the testis, thereby avoiding androgen-induced down-regulation of steroidogenic enzymes, while conversion to 17߭hydroxylated, potent androgens would occur in extra-gonadal tissues. After spawning, pituitary gonadotropin expression and androgens plasma levels decrease to basal levels, and only non-proliferating type A spermatogonia are present as germ cell population in the testis, conditions characterizing the spent condition. Like in many other teleost fish living at high latitudes, seasonally changing parameters are crucial environmental cues for cod. In this context, the present thesis also provides important information on the effects of constant light, a commonly used tool to prevent gonad maturation in cod farms, on the activity of the pituitary-testis axis. For instance, we found that even under constant light conditions, some males can escape the light-mediated inhibition and develop, associated with elevated pituitary lh and testicular ar expressions, spermatogenic activity including sperm production, albeit in a qualitatively and quantitatively compromised manner.

Puberty is the process by which a juvenile acquires for the first time the capacity to reproduce sexually, a critical basis for the conservation of a species. In general, the biological processes constituting the first gonadal maturation and the mechanisms involved in this process are similar among vertebrates. Internal and/or external signals trigger the secretion of gonadotropin-releasing hormone (Gnrh) by neurosecretory neurons in the ventral forebrain, and Gnrh stimulates the synthesis and secretion of two pituitary hormones, the gonadotropins known as follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). These two glycoproteins are the major hormones regulating the remarkable structural and functional changes occurring in the pubertal gonads during the activation of the two principal gonad tasks, the production of gametes and sex steroids. In context with the male-biased problems of precocious testis maturation in farmed cod, the present thesis focuses on male puberty. Pituitary fsh gene expression starts to increase at the beginning of testis development (proliferation of spermatogonia), followed by a slow elevation in testosterone plasma levels. With rapidly continuing spermatogenesis, which takes place in a peculiar spatio-temporal organization in the testicular lobes, high gonado-somatic indexes of up to 16 % are attained. Close to spawning, testosterone and 11-ketotestosterone, two potent ligands for the cod androgen receptor (Ar), peak in parallel with pituitary lh and testicular lhr (Lh receptor) gene expression, while fshr seems to be expressed in a constitutively manner in the cod testis. It is possible that attaining high plasma levels of potent androgens involves an auto-protective manner of androgen production, where weak Ar ligands, such as androstenedione and 11-ketoandrostenedione, are produced by the testis, thereby avoiding androgen-induced down-regulation of steroidogenic enzymes, while conversion to 17߭hydroxylated, potent androgens would occur in extra-gonadal tissues. After spawning, pituitary gonadotropin expression and androgens plasma levels decrease to basal levels, and only non-proliferating type A spermatogonia are present as germ cell population in the testis, conditions characterizing the spent condition. Like in many other teleost fish living at high latitudes, seasonally changing parameters are crucial environmental cues for cod. In this context, the present thesis also provides important information on the effects of constant light, a commonly used tool to prevent gonad maturation in cod farms, on the activity of the pituitary-testis axis. For instance, we found that even under constant light conditions, some males can escape the light-mediated inhibition and develop, associated with elevated pituitary lh and testicular ar expressions, spermatogenic activity including sperm production, albeit in a qualitatively and quantitatively compromised manner.

In the testis a complex process, called spermatogenesis, generates millions of spermatozoa per day. At the start of this process there are spermatogonial stem cells (SSCs) that have the ability to divide either into new stem cells (self-renewal) or daughter cells committed to develop into spermatozoa (differentiation). SSCs are the only cells among the adult stem cell systems capable of transmitting genetic information to future generations. This offers possibilities for in vitro SSC manipulation for instance with the goal of transferring relevant genes across bovine herds. As bovine species have a long generation interval, a system through which genes could be delivered to cattle through SSCs would prove effective and time-saving. SSCs and the first generations of differentiating spermatogonia are morphologically indistinguishable and are called type A spermatogonia. Therefore, we started with finding out at which age type A spermatogonia could be best isolated. We first investigated the onset of spermatogenesis in Brahman bulls, a widely used breed of Asian origin, and compared our results with the available information on European breeds. The best time to obtain A spermatogonia from European and Asian breeds was found to be the age period between the start of spermatogenesis (appearance of the first spermatogonia) to the age at which the first spermatocytes are formed. During this period the purity of the spermatogonia is optimal since no other differentiating germ cells have yet appeared. We then developed a culture system to propagate these A spermatogonia. Using StemPro medium and adding a number of growth factors we could successfully culture the cells for weeks. The usefulness of adding the growth factors GDNF, EGF, LIF and FGF2 was studied. Each of these growth factors was found to exert a specific effect on the culture. EGF, LIF and FGF2 specifically affected the somatic cells in culture while GDNF had a pronounced effect on the numbers of single type A spermatogonia, presumably SSCs. Overall the best results were obtained when the culture medium contained all four growth factors. A 365-fold increase in the numbers of A spermatogonia was found after 4 weeks of culture. To proof that GDNF actually stimulated the propagation of SSCs, a spermatogonial stem cell transplantation assay was carried out in which the cultured cells were transplanted into recipient mouse testes. The transplanted bovine SSCs colonized these testes and the extent of the colonization was taken as a measure of the numbers of bovine SSCs transplanted. A 10.000–fold increase in SSC numbers was found after a culture of 4.5 weeks. During culture in a liquid medium, many germ cells are lost during medium refreshments. To prevent this cell loss, we tested a semisolid culture medium (agar). Indeed, with an agar medium four times as many bovine spermatogonial colonies were able to grow. This indicates that it will be worthwhile to study the advantages of using an agar medium in further detail. In conclusion, we have developed a culture system for the propagation of bovine SSC which will allow further experimentation/manipulation and will eventually lead to more efficient ways of genetical improvement of cattle populations.

In the testis a complex process, called spermatogenesis, generates millions of spermatozoa per day. At the start of this process there are spermatogonial stem cells (SSCs) that have the ability to divide either into new stem cells (self-renewal) or daughter cells committed to develop into spermatozoa (differentiation). SSCs are the only cells among the adult stem cell systems capable of transmitting genetic information to future generations. This offers possibilities for in vitro SSC manipulation for instance with the goal of transferring relevant genes across bovine herds. As bovine species have a long generation interval, a system through which genes could be delivered to cattle through SSCs would prove effective and time-saving. SSCs and the first generations of differentiating spermatogonia are morphologically indistinguishable and are called type A spermatogonia. Therefore, we started with finding out at which age type A spermatogonia could be best isolated. We first investigated the onset of spermatogenesis in Brahman bulls, a widely used breed of Asian origin, and compared our results with the available information on European breeds. The best time to obtain A spermatogonia from European and Asian breeds was found to be the age period between the start of spermatogenesis (appearance of the first spermatogonia) to the age at which the first spermatocytes are formed. During this period the purity of the spermatogonia is optimal since no other differentiating germ cells have yet appeared. We then developed a culture system to propagate these A spermatogonia. Using StemPro medium and adding a number of growth factors we could successfully culture the cells for weeks. The usefulness of adding the growth factors GDNF, EGF, LIF and FGF2 was studied. Each of these growth factors was found to exert a specific effect on the culture. EGF, LIF and FGF2 specifically affected the somatic cells in culture while GDNF had a pronounced effect on the numbers of single type A spermatogonia, presumably SSCs. Overall the best results were obtained when the culture medium contained all four growth factors. A 365-fold increase in the numbers of A spermatogonia was found after 4 weeks of culture. To proof that GDNF actually stimulated the propagation of SSCs, a spermatogonial stem cell transplantation assay was carried out in which the cultured cells were transplanted into recipient mouse testes. The transplanted bovine SSCs colonized these testes and the extent of the colonization was taken as a measure of the numbers of bovine SSCs transplanted. A 10.000–fold increase in SSC numbers was found after a culture of 4.5 weeks. During culture in a liquid medium, many germ cells are lost during medium refreshments. To prevent this cell loss, we tested a semisolid culture medium (agar). Indeed, with an agar medium four times as many bovine spermatogonial colonies were able to grow. This indicates that it will be worthwhile to study the advantages of using an agar medium in further detail. In conclusion, we have developed a culture system for the propagation of bovine SSC which will allow further experimentation/manipulation and will eventually lead to more efficient ways of genetical improvement of cattle populations.

In the testis a complex process, called spermatogenesis, generates millions of spermatozoa per day. At the start of this process there are spermatogonial stem cells (SSCs) that have the ability to divide either into new stem cells (self-renewal) or daughter cells committed to develop into spermatozoa (differentiation). SSCs are the only cells among the adult stem cell systems capable of transmitting genetic information to future generations. This offers possibilities for in vitro SSC manipulation for instance with the goal of transferring relevant genes across bovine herds. As bovine species have a long generation interval, a system through which genes could be delivered to cattle through SSCs would prove effective and time-saving. SSCs and the first generations of differentiating spermatogonia are morphologically indistinguishable and are called type A spermatogonia. Therefore, we started with finding out at which age type A spermatogonia could be best isolated. We first investigated the onset of spermatogenesis in Brahman bulls, a widely used breed of Asian origin, and compared our results with the available information on European breeds. The best time to obtain A spermatogonia from European and Asian breeds was found to be the age period between the start of spermatogenesis (appearance of the first spermatogonia) to the age at which the first spermatocytes are formed. During this period the purity of the spermatogonia is optimal since no other differentiating germ cells have yet appeared. We then developed a culture system to propagate these A spermatogonia. Using StemPro medium and adding a number of growth factors we could successfully culture the cells for weeks. The usefulness of adding the growth factors GDNF, EGF, LIF and FGF2 was studied. Each of these growth factors was found to exert a specific effect on the culture. EGF, LIF and FGF2 specifically affected the somatic cells in culture while GDNF had a pronounced effect on the numbers of single type A spermatogonia, presumably SSCs. Overall the best results were obtained when the culture medium contained all four growth factors. A 365-fold increase in the numbers of A spermatogonia was found after 4 weeks of culture. To proof that GDNF actually stimulated the propagation of SSCs, a spermatogonial stem cell transplantation assay was carried out in which the cultured cells were transplanted into recipient mouse testes. The transplanted bovine SSCs colonized these testes and the extent of the colonization was taken as a measure of the numbers of bovine SSCs transplanted. A 10.000–fold increase in SSC numbers was found after a culture of 4.5 weeks. During culture in a liquid medium, many germ cells are lost during medium refreshments. To prevent this cell loss, we tested a semisolid culture medium (agar). Indeed, with an agar medium four times as many bovine spermatogonial colonies were able to grow. This indicates that it will be worthwhile to study the advantages of using an agar medium in further detail. In conclusion, we have developed a culture system for the propagation of bovine SSC which will allow further experimentation/manipulation and will eventually lead to more efficient ways of genetical improvement of cattle populations.

DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatids. Spermatogonia include stem cells and subsequent types of proliferating spermatogonia and express many DNA repair proteins including gamma-H2AX (a marker for DSBs) and p53 binding protein1 (53BP1) but not MDC1 (mediator of DNA damage checkpoint). MDC1 is an important protein that functions in both HR and NHEJ. In the lack of MDC1, spermatogonia differ from proliferating somatic cells, and this may explain why our studies revealed a slow and incomplete repair of DSBs in spermatogonia. In early spermatocytes (leptotene, zygotene and early pachytene stages of the meiotic prophase) during meiotic recombination DSB are induced. Our findings indicate that, the error prone repair via NHEJ does not take place in early spermatocytes, also not to repair radiation induced DSBs. First, these cells do not express 53BP1, an important protein in NHEJ. Second, this notion could be further established by studying the repair of recombination related DSBs and of DSBs induced by irradiation in scid mice (deficient for NHEJ) and in Rad54/Rad54B deficient mice, potentially deficient for HR. Repair of DSBs in early spermatocytes in scid mice was similar to that in wild type mice, however the repair was significantly lower in Rad54/Rad54B deficient mice. Hence, DSB repair in early spermatocytes is exclusively carried out through HR. In late spermatocytes (mid-pachytene through diplotene stages) all important repair proteins were found and using Rad54/Rad54B deficient mice, and scid mice, we could establish that both NHEJ and HR pathways are active in these cells, NHEJ probably being the most important. The meiotic divisions render round spermatids that elongate and transform into spermatozoa. In spermatids, HR is not possible as thy are haploid and earlier results indicate that in these cells DNA-PKcs, a protein indispensable for classical NHEJ, is not expressed or at very low levels. Nevertheless, after irradiation gamma-H2AX foci disappear in these cells indicating that these cells are capable of DSB repair. Using scid mice and mice in which PARP1 protein function was inhibited, we could establish that in spermatids some residual classical NHEJ takes place and that about half of the repair takes place through the recently discovered alternative pathway for NHEJ, involving the PARP1/XRCC1 proteins. Nevertheless, DSB repair in round spermatids is slow and incomplete. We also found that, in contrast to other somatic cells in the testis, Sertoli cells, the supporting somatic cells in the seminiferous epithelium, express several DNA repair proteins and repair DSBs, as shown by the in vivo disappearance of 53BP1 foci after irradiation and the results of the comet assay. This, together with our finding that adult Sertoli cells resume proliferation in culture indicates that adult Sertoli cells can no longer be regarded as a classical example of terminally differentiated cells that are no longer able to proliferate and do not repair DNA DSBs.

DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatids. Spermatogonia include stem cells and subsequent types of proliferating spermatogonia and express many DNA repair proteins including gamma-H2AX (a marker for DSBs) and p53 binding protein1 (53BP1) but not MDC1 (mediator of DNA damage checkpoint). MDC1 is an important protein that functions in both HR and NHEJ. In the lack of MDC1, spermatogonia differ from proliferating somatic cells, and this may explain why our studies revealed a slow and incomplete repair of DSBs in spermatogonia. In early spermatocytes (leptotene, zygotene and early pachytene stages of the meiotic prophase) during meiotic recombination DSB are induced. Our findings indicate that, the error prone repair via NHEJ does not take place in early spermatocytes, also not to repair radiation induced DSBs. First, these cells do not express 53BP1, an important protein in NHEJ. Second, this notion could be further established by studying the repair of recombination related DSBs and of DSBs induced by irradiation in scid mice (deficient for NHEJ) and in Rad54/Rad54B deficient mice, potentially deficient for HR. Repair of DSBs in early spermatocytes in scid mice was similar to that in wild type mice, however the repair was significantly lower in Rad54/Rad54B deficient mice. Hence, DSB repair in early spermatocytes is exclusively carried out through HR. In late spermatocytes (mid-pachytene through diplotene stages) all important repair proteins were found and using Rad54/Rad54B deficient mice, and scid mice, we could establish that both NHEJ and HR pathways are active in these cells, NHEJ probably being the most important. The meiotic divisions render round spermatids that elongate and transform into spermatozoa. In spermatids, HR is not possible as thy are haploid and earlier results indicate that in these cells DNA-PKcs, a protein indispensable for classical NHEJ, is not expressed or at very low levels. Nevertheless, after irradiation gamma-H2AX foci disappear in these cells indicating that these cells are capable of DSB repair. Using scid mice and mice in which PARP1 protein function was inhibited, we could establish that in spermatids some residual classical NHEJ takes place and that about half of the repair takes place through the recently discovered alternative pathway for NHEJ, involving the PARP1/XRCC1 proteins. Nevertheless, DSB repair in round spermatids is slow and incomplete. We also found that, in contrast to other somatic cells in the testis, Sertoli cells, the supporting somatic cells in the seminiferous epithelium, express several DNA repair proteins and repair DSBs, as shown by the in vivo disappearance of 53BP1 foci after irradiation and the results of the comet assay. This, together with our finding that adult Sertoli cells resume proliferation in culture indicates that adult Sertoli cells can no longer be regarded as a classical example of terminally differentiated cells that are no longer able to proliferate and do not repair DNA DSBs.

BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.

It was previously shown that administration of recombinant human Fms-like tyrosine kinase receptor-3 ligand (Flt3L) before allergen challenge of sensitized mice suppresses the cardinal features of asthma through unclear mechanisms. Here, we show that Flt3L dramatically alters the balance of conventional to plasmacytoid dendritic cells (pDCs) in the lung favoring the accumulation of pDCs. Selective removal of pDCs abolished the antiinflammatory effect of Flt3L, suggesting a regulatory role for these cells in ongoing asthmatic inflammation. In support, we found that immature pDCs are recruited to the lungs of allergen-challenged mice irrespective of Flt3L treatment. Selective removal of pDCs during allergen challenge enhanced airway inflammation, whereas adoptive transfer of cultured pDCs before allergen challenge suppressed inflammation. Experiments in which TLR9 agonist CpG motifs were administered in vitro or in vivo demonstrated that pDCs were antiinflammatory irrespective of their maturation state. These effects were mediated through programmed death-1/programmed death ligand 1 interactions, but not through ICOS ligand, IDO, or IFN-alpha. These findings suggest a specialized immunoregulatory role for pDCs in airway inflammation. Enhancing the antiinflammatory properties of pDCs could be employed as a novel strategy in asthma treatment.

It was previously shown that administration of recombinant human Fms-like tyrosine kinase receptor-3 ligand (Flt3L) before allergen challenge of sensitized mice suppresses the cardinal features of asthma through unclear mechanisms. Here, we show that Flt3L dramatically alters the balance of conventional to plasmacytoid dendritic cells (pDCs) in the lung favoring the accumulation of pDCs. Selective removal of pDCs abolished the antiinflammatory effect of Flt3L, suggesting a regulatory role for these cells in ongoing asthmatic inflammation. In support, we found that immature pDCs are recruited to the lungs of allergen-challenged mice irrespective of Flt3L treatment. Selective removal of pDCs during allergen challenge enhanced airway inflammation, whereas adoptive transfer of cultured pDCs before allergen challenge suppressed inflammation. Experiments in which TLR9 agonist CpG motifs were administered in vitro or in vivo demonstrated that pDCs were antiinflammatory irrespective of their maturation state. These effects were mediated through programmed death-1/programmed death ligand 1 interactions, but not through ICOS ligand, IDO, or IFN-alpha. These findings suggest a specialized immunoregulatory role for pDCs in airway inflammation. Enhancing the antiinflammatory properties of pDCs could be employed as a novel strategy in asthma treatment.

Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged. Furthermore, our results suggest that superlattice nucleation and growth occurred at the suspension/air interface and that the unit cells of some lattices are anisotropically deformed upon drying.

Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged. Furthermore, our results suggest that superlattice nucleation and growth occurred at the suspension/air interface and that the unit cells of some lattices are anisotropically deformed upon drying.

When observing bistable stimuli, the percept can change in the absence of changes in the stimulus itself. When intermittently presenting a bistable stimulus, the number of perceptual alternations can increase or decrease, depending on the duration of the period that the stimulus is removed from screen between stimulus presentations (off-period). Longer off-periods lead to stabilization of the percept, while short off-periods produce perceptual alternations. Here we compare fMRI brain activation across percept repetitions and alternations when observing an intermittently presented ambiguously rotating structure from motion sphere. In the first experimental session, subjects were requested to voluntarily control the percept into either a repeating or an alternating perceptual regime at a single off-period. In a consecutive session, subjects observed the sphere uninstructed, and reported alternations and repetitions. The behavioral data showed that there were marked individual biases for observing the sphere as either repeating or alternating. The fMRI data showed activation differences between alternating and repeating perceptual regimes in an extensive network that included parietal cortex, dorsal premotor area, dorsolateral prefrontal cortex, supplementary motor area, insula, and cerebellum. However, these activation differences could all be explained by intersubject differences in the bias for one of the two perceptual regimes. The stronger the bias was for a particular perceptual regime, the less activation and vice versa. We conclude that widespread activation differences between perceptual regimes can be accounted for by differences in the perceptual bias for one of the two regimes

In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10-4-10-1 M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure.

When observing bistable stimuli, the percept can change in the absence of changes in the stimulus itself. When intermittently presenting a bistable stimulus, the number of perceptual alternations can increase or decrease, depending on the duration of the period that the stimulus is removed from screen between stimulus presentations (off-period). Longer off-periods lead to stabilization of the percept, while short off-periods produce perceptual alternations. Here we compare fMRI brain activation across percept repetitions and alternations when observing an intermittently presented ambiguously rotating structure from motion sphere. In the first experimental session, subjects were requested to voluntarily control the percept into either a repeating or an alternating perceptual regime at a single off-period. In a consecutive session, subjects observed the sphere uninstructed, and reported alternations and repetitions. The behavioral data showed that there were marked individual biases for observing the sphere as either repeating or alternating. The fMRI data showed activation differences between alternating and repeating perceptual regimes in an extensive network that included parietal cortex, dorsal premotor area, dorsolateral prefrontal cortex, supplementary motor area, insula, and cerebellum. However, these activation differences could all be explained by intersubject differences in the bias for one of the two perceptual regimes. The stronger the bias was for a particular perceptual regime, the less activation and vice versa. We conclude that widespread activation differences between perceptual regimes can be accounted for by differences in the perceptual bias for one of the two regimes

In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10-4-10-1 M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure.

In tropical lowland forests, which dominated by trees of Dipterocarpaceae family, trees and fungi are strongly linked, through ectomycorrhiza (EcM) formation. However, as part of rapid deforestation and forest transformation, mixed dipterocarp forests have been replaced by other vegetation and land use types. Considerable areas of the lowland tropical forest in Sumatra have been turned into Rubber Agroforests (RAF). RAF is a land use type in which rubber trees are planted, while allowing spontaneous establishment of forest tree species. The method presented in this thesis includes four main elements. Firstly, vegetation analysis in seven types of land use. Secondly, study in a nursery using dipterocarps species as bait plant to assess the effect of land use change on EcM inoculum and to analyze the effect of soil heating and drying on EcM propagules. Thirdly, an in-situ experiment in RAF with different history was done to assess whether fungal inoculant increased the survival, growth, nutrient uptake and EcM formation on Shorea selanica and Shorea lamellata. Finally, molecular techniques of PCR and sequencing have been applied to identify EcM fungal symbionts on dipterocarps. The study has been conducted in Jambi province, Sumatra, Indonesia. The ex-situ experiment has been conducted in the nursery of the Forest Research and Development Agency, Bogor, Indonesia. Identification of EcM fungi was done in the Fungal Biodiversity Centre (CBS), Utrecht, the Netherlands. The result of vegetation analysis showed that human activity in RAF management has strong effects on species richness. Very few trees dependent on EcM fungi were encountered in RAF. Our study in the nursery shows small differences in EcM colonization between soils derived from a wide range of land use types. It may indicate that survival of spores and colonization potential are not sensitive to the history of the site. Rapid recolonization of the heated soil in the experiment may imply sufficient availability of EcM fungi for dipterocarps under field conditions, where fire as a tool for land clearing is commonly used in Sumatra. The in-situ experiment in the RAF showed that lack of trees that are dependent on EcM fungi in RAF and in non-forested land does not necessarily imply the absence of EcM inoculum belowground in the ecosystem. EcM inoculum persists in the soil after forest was changed to RAF. Molecular identification of EcM fungi showed that Tomentella was the prevalent genus of EcM fungi colonizing dipterocarp seedlings in nursery stage and in the in-situ test 1 year after the seedlings have been planted in the field. None of the fungi was identified as Scleroderma columnare, which was inoculated to S. lamellata and S. selanica seedlings in the nursery stage. This indicated that indigenous EcM fungi dominated on the dipterocarp seedlings planted in the field, regardless of nursery inoculation. The implementation of this study is potentially of great value for reforestation efforts with dipterocarps in RAF in collaboration with rubber farmers. This reforestation approach will ensure the successfulness of the reforestation managed sustainably by the farmer, from which they may benefit in the future.