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Anthropogenic activities have altered historical disturbance regimes, and understanding the mechanisms by which these shifting perturbations interact is essential to predicting where they may erode ecosystem resilience. Emerging infectious plant diseases, caused by human translocation of nonnative pathogens, can generate ecologically damaging forms of novel biotic disturbance. Further, abiotic disturbances, such as wildfire, may influence the severity and extent of disease‐related perturbations via their effects on the occurrence of hosts, pathogens and microclimates; however, these interactions have rarely been examined. The disease ‘sudden oak death’ (SOD), associated with the introduced pathogen Phytophthora ramorum, causes acute, landscape‐scale tree mortality in California's fire‐prone coastal forests. Here, we examined interactions between wildfire and the biotic disturbance impacts of this emerging infectious disease. Leveraging long‐term datasets that describe wildfire occurrence and P. ramorum dynamics across the Big Sur region, we modelled the influence of recent and historical fires on epidemiological parameters, including pathogen presence, infestation intensity, reinvasion, and host mortality. Past wildfire altered disease dynamics and reduced SOD‐related mortality, indicating a negative interaction between these abiotic and biotic disturbances. Frequently burned forests were less likely to be invaded by P. ramorum, had lower incidence of host infection, and exhibited decreased disease‐related biotic disturbance, which was associated with reduced occurrence and density of epidemiologically significant hosts. Following a recent wildfire, survival of mature bay laurel, a key sporulating host, was the primary driver of P. ramorum infestation and reinvasion, but younger, rapidly regenerating host vegetation capable of sporulation did not measurably influence disease dynamics. Notably, the effect of P. ramorum infection on host mortality was reduced in recently burned areas, indicating that the loss of tall, mature host canopies may temporarily dampen pathogen transmission and ‘release’ susceptible species from significant inoculum pressure. Synthesis. Cumulatively, our findings indicate that fire history has contributed to heterogeneous patterns of biotic disturbance and disease‐related decline across this landscape, via changes to the both the occurrence of available hosts and the demography of epidemiologically important host populations. These results highlight that human‐altered abiotic disturbances may play a foundational role in structuring infectious disease dynamics, contributing to future outbreak emergence and driving biotic disturbance regimes.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. (C) 2021 American Society for Bone and Mineral Research. (C) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Plant defense chemistry is often hypothesized to drive ecological and evolutionary success in diverse tropical forests, yet detailed characterizations of plant secondary metabolites in tropical plants are logistically challenging. Here, we explore a new integrative approach that combines visible‐to‐shortwave infrared (VSWIR) spectral reflectance data with detailed plant metabolomics data from 19 Protium (Burseraceae) tree species. Building on the discovery that different Protium species have unique chemistries yet share many secondary metabolites, we devised a method to test for associations between metabolites and VSWIR spectral data. Given species‐level variation in metabolite abundance, we correlated the concentration of particular chemicals with the reflectance of the spectral bands in a wavelength band per secondary metabolite matrix. We included 45 metabolites that were shared by at least 5 Protium species and correlated their per‐species foliar abundances against each one of 210 wavelength bands of field‐measured VSWIR spectra. Finally, we tested whether classes of similar metabolites showed similar relationships with spectral patterns. We found that many secondary metabolites yielded strong correlations with VSWIR spectra of Protium. Furthermore, important Protium metabolite classes such as procyanidins (condensed tannins) and phytosterols were grouped together in a hierarchical clustering analysis (Ward’s algorithm), confirming similarity in their associations with plant spectral patterns. We also found a significant correlation in the phenolics content between juvenile and canopy trees of the same species, suggesting that species‐level variation in defense chemistry is consistent across life stages and geographic distribution. We conclude that the integration of spectral and metabolic approaches could represent a powerful and economical method to characterize important aspects of tropical plant defense chemistry.

The zebrafish is an appealing model organism for investigating the genetic (G) and environmental (E) factors, as well as their interactions (GxE), which contribute to craniofacial malformations. Here, we review zebrafish studies on environmental factors involved in the etiology of craniofacial malformations in humans including maternal smoking, alcohol consumption, nutrition and drug use. As an example, we focus on the (cleft) palate, for which the zebrafish ethmoid plate is a good model. This review highlights the importance of investigating ExE interactions and discusses the variable effects of exposure to environmental factors on craniofacial development depending on dosage, exposure time and developmental stage. Zebrafish also promise to be a good tool to study novel craniofacial teratogens and toxin mixtures. Lastly, we discuss the handful of studies on gene–alcohol interactions using mutant sensitivity screens and reverse genetic techniques. We expect that studies addressing complex interactions (ExE and GxE) in craniofacial malformations will increase in the coming years. These are likely to uncover currently unknown mechanisms with implications for the prevention of craniofacial malformations. The zebrafish appears to be an excellent complementary model with high translational value to study these complex interactions.

Zebrafish scales are mineralised plates that can regenerate involvingde novobone formation. This presents an opportunity to uncover genes and pathways relevant to human musculoskeletal disease relevant to impaired bone formation. To investigate this hypothesis, we defined transcriptomic profiles of ontogenetic and regenerating scales, and identified 604 differentially expressed genes (DEGs) that were enriched for extracellular matrix, ossification, and cell adhesion pathways. Next, we showed that human orthologues of DEGs were 2.8 times more likely to cause human monogenic skeletal diseases (P−11), and they showed enrichment for human orthologues associated with polygenetic disease traits including stature, bone density and osteoarthritis (PCOL11A2) or bone density only (SPP1) developed skeletal abnormalities consistent with our genetic association studies.Col11a2Y228X/Y228Xmutants showed endoskeletal features consistent with abnormal growth and osteoarthritis, whereasspp1P160X/P160Xmutants had elevated bone density (P

The tropical forests of Borneo and Amazonia may each contain more tree species diversity in half a square kilometre than do all the temperate forests of Europe, North America, and Asia combined. Biologists have long been fascinated by this disparity, using it to investigate potential drivers of biodiversity. Latitudinal variation in many of these drivers is expected to create geographic differences in ecological and evolutionary processes, and evidence increasingly shows that tropical ecosystems have higher rates of diversification, clade origination, and clade dispersal. However, there is currently no evidence to link gradients in ecological processes within communities at a local scale directly to the geographic gradient in biodiversity. Here, we show geographic variation in the storage effect, an ecological mechanism that reduces the potential for competitive exclusion more strongly in the tropics than it does in temperate and boreal zones, decreasing the ratio of interspecific-to-intraspecific competition by 0.25% for each degree of latitude that an ecosystem is located closer to the Equator. Additionally, we find evidence that latitudinal variation in climate underpins these differences; longer growing seasons in the tropics reduce constraints on the seasonal timing of reproduction, permitting lower recruitment synchrony between species and thereby enhancing niche partitioning through the storage effect. Our results demonstrate that the strength of the storage effect, and therefore its impact on diversity within communities, varies latitudinally in association with climate. This finding highlights the importance of biotic interactions in shaping geographic diversity patterns, and emphasizes the need to understand the mechanisms underpinning ecological processes in greater detail than has previously been appreciated.

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 μM VPA), the posterior head length and the ethmoid plate were reduced in length (both p 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p 0.01). In the late group (100 μM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

Human‐altered disturbance regimes and changing climatic conditions can reduce seed availability and suitable microsites, limiting seedling regeneration in recovering forest systems. Thus, resprouting plants, which can persist in situ, are expected to expand in dominance in many disturbance‐prone forests. However, resprouters may also be challenged by changing regimes, and the mechanisms determining facultative seedling recruitment by resprouting species, which will determine both the future spread and current persistence of these populations, are poorly understood. In the resprouter‐dominated forests of coastal California, interactions between wildfire and an emerging disease, sudden oak death (SOD), alter disturbance severity and tree mortality, which may shift forest regeneration trajectories. We examine this set of compound disturbances to (1) assess the influence of seed limitation, biotic competition, and abiotic conditions on seedling regeneration in resprouting populations; (2) investigate whether disease‐fire interactions alter postfire seedling regeneration, which have implications for future disease dynamics and shifts in forest composition. Following a wildfire that impacted a preexisting plot network in SOD‐affected forests, we monitored seedling abundances and survival over eight years. With pre‐ and postfire data, we assessed relationships between regeneration dynamics and disturbance severity, biotic, and abiotic variables, using Bayesian generalized linear models and mixed models. Our results indicate that postfire seedling regeneration by resprouting species was shaped by contrasting mechanisms reflecting seed limitation and competitive release. Seedling abundances declined with decreasing postfire survival of mature, conspecific stems, while belowground survival of resprouting genets had no effect. However, where seed sources persisted, seedling abundances and survival generally increased with the prefire severity of disease impacts, suggesting that decreased competition with adults may enhance seedling recruitment in this resprouter‐dominated system. Species’ regeneration responses varied with their relative susceptibility to SOD and suggest compositional shifts, which will determine future disease management and forest restoration actions. These results additionally highlight that mechanisms related to biotic competition, seed limitation, and opportunities for seedling recruitment beneath mature canopies may determine possible shifts in the occurrence of resprouting traits. This result has broad applications to other systems impacted by human‐altered regimes where asexual persistence may be predicted to be a beneficial life history strategy.

The global mechanisms that regulate and potentially coordinate cell proliferation & death in developing neural regions are not well understood. In particular, it is not clear how or whether clonal relationships between neural progenitor cells and their progeny influence the growing brain. We have developed an approach using Brainbow in the developing zebrafish to visualize and follow multiple clones of related cells in vivo over time. This allows for clear visualization of many dividing clones of cells, deep in proliferating brain regions. As expected, in addition to undergoing interkinetic nuclear migration and cell division, cells also periodically undergo apoptosis. Interestingly, cell death occurs in a non-random manner: clonally related cells are more likely to die in a progressive fashion than cells from different clones. Multiple members of an individual clone die while neighboring clones appear healthy and continue to divide. Our results suggest that clonal relationships can influence cellular fitness and survival in the developing nervous system, perhaps through a competitive mechanism whereby clones of cells are competing with other clones. Clonal cell competition may help regulate neuronal proliferation in the vertebrate brain.

Craniofacial development is tightly regulated and therefore highly vulnerable to disturbance by genetic and environmental factors. Fibroblast growth factors (FGFs) direct migration, proliferation and survival of cranial neural crest cells (CNCCs) forming the human face. In this study, we analyzed bone and cartilage formation in the head of five dpf fgf8ati282 zebrafish larvae and assessed gene expression levels for 11 genes involved in these processes. In addition, in situ hybridization was performed on 8 and 24 hours post fertilization (hpf) larvae (fgf8a, dlx2a, runx2a, col2a1a). A significant size reduction of eight out of nine craniofacial cartilage structures was found in homozygous mutant (6–36%, P, Summary: A function-blocking mutation in fgf8a causes craniofacial malformations in zebrafish larvae due to impaired cranial neural crest cell migration and survival.

Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.

This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX-induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0-5 hours) of wound-healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up-regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life.

In Amazonian Peru, the neotropical tree Protium subserratum Engl. (Burseraceae) occurs as distinct ecotypes on low nutrient white-sand (WS), intermediate fertility brown-sand (BS), and nutrient-rich clay (CS) soils. Genetic analysis indicates that these ecotypes are undergoing incipient speciation. Possible drivers of this divergence are habitat-specific herbivore faunas and differing resource availabilities. Protium subserratum, therefore, provides an ideal opportunity to investigate how defense chemistry evolves during lineage divergence. WS and BS races of P. subserratum are host to largely non-overlapping herbivore communities and they differ in chlorogenic acid, flavonoid, and oxidized terpene chemistry. Here, we investigate how another important class of anti-herbivore chemicals, procyanidins (PCs), varies among the ecotypes. We isolated the PCs from leaves of juvenile and adult trees from each ecotype and used spectroscopic and chemical techniques to characterize the chemical structures of their component monomers. We found that WS, BS, and CS ecotypes accumulate ca. 17 % of leaf dry weight as PCs. Within ecotypes, we found very little difference in PC type, neither by site nor by life stage. Among ecotypes, however, we observed a marked divergence in PC composition that arose at least in part from differences in their terminal and extension subunits. In addition, the average polymer length of BS and CS PCs was significantly greater than in WS ecotypes. We conclude that phenotypic differences in PCs in the WS versus BS and CS ecotypes of P. subserratum are consistent with selection by herbivores in different soil types that differ strongly in nutrient availability and may contribute to the evolution of habitat specialization.

Human-altered ecological disturbances may challenge system resilience and disrupt biological legacies maintaining ecosystem recovery. Yet, the extent to which novel regimes challenge these legacies varies. This may be partially explained by differences in the vulnerability of life history strategies to disturbance characteristics. In the fire-prone, resprouter-dominated coast redwood forests of California, the introduced disease sudden oak death (SOD) alters fuel profiles, fire behavior, and aboveground tree mortality; however, this system is dominated by resprouting trees that are well-adapted to aboveground damage, and belowground survival of individuals may represent the principal biological legacy connecting pre- and post-fire communities. Much of the research exploring altered disturbances and forest recovery has focused on legacies determined by seed dispersal and aboveground survival of adults. In this work, we use pre- and post-fire data from a long-term monitoring network to assess the impacts of novel disturbance interactions between wildfire and SOD on the belowground survival and vegetative reproduction of resprouters. We found that increasing accumulation of coarse woody surface fuels from SOD-killed hosts decreased the likelihood of belowground survival for resprouting tanoak trees, but not for redwoods. Tanoaks' belowground survival was negatively related to substrate burn severity, which increased with the volume of surface fuels from hosts, suggesting heat damage as a possible mechanism influencing altered patterns of resprouter mortality. These impacts increased with decreasing tree size. By contrast, redwood and tanoak trees that survived both disturbances resprouted more vigorously, regardless of post-fire infection by P. ramorum, and generated similar recruitment at the stand level. Our results demonstrate that disease-fire interactions can narrow recruitment filters for resprouters, which could impact long-term population and demographic structure; yet, compounded disturbance may also reduce stand density and disease pressure, allowing competitive release of survivors. Resprouters displayed vulnerabilities to altered disturbance, but our research suggests that legacies maintained by resprouting may be more resilient to certain compounded disturbances, compared to seed-obligate species, because of high rates of individual survival under increasingly severe events. These trends have important implications for conservation of declining tree species in SOD-impacted forests, as well as predictions of human impacts in other disturbance-prone systems where resprouters are present.

Environmental contaminants have previously been demonstrated to cause bone deformities mediated through the aryl hydrocarbon receptor (AhR) in fish and mammals. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment, many of them capable of activating AhR. In the present study, fish scales were utilized as a model system to examine possible AhR‐mediated effects of PAHs on bone forming osteoblasts and bone resorptive osteoclasts, using the AhR‐ligand benzo(a)pyrene (BaP) as a model compound. Elasmoid scales from goldfish and zebrafish were exposed to 0.005–50 μM BaP for up to 48 hr, and the activity of osteoblastic and osteoclastic markers were measured, as well as mRNA levels of bone related genes and cyp1a and cyp3a. Using the sp7:luciferase zebrafish assay, a decrease in sp7 promoter activation was observed at the two highest concentrations (5 and 50 μM). Gelatin zymography revealed significantly reduced activity of the osteoclastic protease matrix metalloproteinase 9 (Mmp9) at the highest concentration. Furthermore, transcriptional analysis showed a dose‐dependent increase in cyp1a, however, no significant differential expression was observed for the bone related genes. The findings indicate that BaP might decrease differentiation and activation of osteoblasts, and reduce osteoclastic activity, and thus ultimately cause decreased bone formation. Further investigation is necessary in order to confirm the role of AhR in mediating these effects.

The 2008 wildfires in the Big Sur region of California’s central coast—the first to occur in forests impacted by Phytophthora ramorum, the non-native, invasive pathogen that causes sudden oak death—provided the rare opportunity to study the response of scolytid and other subcortical beetles to this novel disturbance interaction. We used sticky card traps attached to the main stem of tanoak, Notholithocarpus densiflorus, the tree species most susceptible to P. ramorum, to determine which subcortical beetle species may be using tanoak as a host and to compare insect landing rates on these trees in forest plots impacted by neither disturbance, either wildfire or P. ramorum disturbance alone, or both disturbances combined. Xyleborinus saxesenii and Gnathotrichus pilosus, two species of ambrosia beetles (Coleoptera: Scolytidae), composed the majority (48% and 40%, respectively) of subcortical beetles landing on tanoaks during both years of the study. Adults of two species of a small, branch-feeding flatheaded borer (Anthaxia sp.; Coleoptera: Buprestidae) were also captured in relative abundance landing on tanoaks in the combined disturbance plots during the second year of the study. All but two of the 2779 scolytid beetles collected in this study were trapped on tanoaks in forest plots disturbed by P. ramorum and/or fire, and 75% of these scolytids were trapped during the fall 2009 season. The majority of scolytids were trapped on tanoaks in plots containing both disturbances (81% in 2009 and 79% in 2010), and, of the two disturbances, more scolytids were trapped on tanoaks in burned plots than in P. ramorum-infested plots (92% more in 2009 and 476% more in 2010). Semiochemicals emanating from the tanoaks upon which the sticky cards were attached—either in the form of host volatile compounds or scolytid aggregation pheromones—presumably affected ambrosia beetle landing rates, and greater quantities of moribund and recently-killed trees in the plots disturbed both by P. ramorum and fire may have led to greater population densities of ambrosia beetles in these areas. Our findings of elevated ambrosia beetle landing rates in Big Sur forests with mixed disturbances suggest a heightened threat to tanoak in these areas, but additional research is needed to determine the actual frequency of ambrosia beetle gallery initiation in living tanoaks and whether colonization hastens or leads to tree mortality.

BACKGROUND Many tropical forest tree species delay greening their leaves until full expansion. This strategy is thought to provide newly flushing leaves with protection against damage by herbivores by keeping young leaves devoid of nutritive value. Because young leaves suffer the greatest predation from invertebrate herbivores, delayed greening could prevent costly tissue loss. Many species that delay greening also produce anthocyanin pigments in their new leaves, giving them a reddish tint. These anthocyanins may be fungicidal, protect leaves against UV damage or make leaves cryptic to herbivores blind to the red part of the spectrum. METHODS A comprehensive survey was undertaken of seedlings, saplings and mature trees in two diverse tropical forests: a rain forest in western Amazonia (Yasuni National Park, Ecuador) and a deciduous forest in Central America (Barro Colorado Island, Panama). A test was made of whether individuals and species with delayed greening or red-coloured young leaves showed lower mortality or higher relative growth rates than species that did not. KEY RESULTS At both Yasuni and Barro Colorado Island, species with delayed greening or red young leaves comprised significant proportions of the seedling and tree communities. At both sites, significantly lower mortality was found in seedlings and trees with delayed greening and red-coloured young leaves. While there was little effect of leaf colour on the production of new leaves of seedlings, diameter relative growth rates of small trees were lower in species with delayed greening and red-coloured young leaves than in species with regular green leaves, and this effect remained when the trade-off between mortality and growth was accounted for. CONCLUSIONS Herbivores exert strong selection pressure on seedlings for the expression of defence traits. A delayed greening or red-coloured young leaf strategy in seedlings appears to be associated with higher survival for a given growth rate, and may thus influence the species composition of later life stages.

The geographic range of tanoak, Notholithocarpus densiflorus (Hook. & Arn.) Manos, Cannon & S. H. Oh (Fagaceae), encompasses tremendous physiographic variability, diverse plant communities, and complex disturbance regimes (e.g., development, timber harvest, and wildfire) that now also include serious threats posed by the invasive forest pathogen Phytophthora ramorum S. Werres, A.W.A.M. de Cock. Knowing where these disturbance factors interact is critical for developing comprehensive strategies for conserving the abundance, structure, and function of at-risk tanoak communities. In this study, we present a rule-based spatial model of the range-wide threat to tanoak populations from four disturbance factors that were parameterized to encode their additive effects and two-way interactions. Within a GIS, we mapped threats posed by silvicultural activities; disease caused by P. ramorum; human development; and altered fire regimes across the geographic range of tanoak, and we integrated spatially coinciding disturbances to quantify and map the additive and interacting threats to tanoak. We classified the majority of tanoak's range at low risk (3.7 million ha) from disturbance interactions, with smaller areas at intermediate (222,795 ha), and high (10,905 ha) risk. Elevated risk levels resulted from the interaction of disease and silviculture factors over small extents in northern California and southwest Oregon that included parts of Hoopa and Yurok tribal lands. Our results illustrate tanoak populations at risk from these interacting disturbances based on one set of hypothesized relationships. The model can be extended to other species affected by these factors, used as a guide for future research, and is a point of departure for developing a comprehensive understanding of threats to tanoak populations. Identifying the geographic location of disturbance interactions and risks to foundation species such as tanoak is critical for prioritizing and targeting conservation treatments with limited resources.

Human activities and climate change have altered historical disturbance regimes, introduced disturbances, and encouraged novel interactions between multiple disturbances. Ecosystems and the species that comprise them may be poorly equipped to withstand or recover from these altered disturbance regimes. In the fire-prone coastal forests of California and Oregon, sudden oak death (SOD), caused by the pathogen Phytophthora ramorum, is an emerging, non-native plant disease that causes widespread tree mortality and associated implications for fire regimes. Disease-related tree mortality alters fuel loads, with patterns of fuel accumulation varying depending on stand composition, disease severity, and time since pathogen invasion. Simulations and observational studies suggest these altered fuel profiles can impact subsequent fire behavior, and the extent of this interaction may depend on the severity and timing of disease impacts. Initial tree death can elevate the risk of crown ignition, while latter stages can increase surface fuel loading and have been linked to increased fire severity in wildfires. Further, disease history can also influence fire severity with cascading effects leading to unexpected increases in mortality of non-susceptible tree species and changes in nutrient cycling. The longer-term impacts of SOD-fire interactions on system resilience and recovery remain to be seen, but increased fire severity, changed stand structure, and altered biogeochemical cycling may have important consequences for post-fire regeneration and future ecosystem function. Fuels management strategies that diminish crown fire hazards at early stages and mitigate surface fuel hazards at later stages offer some promise, but have yet to be tested in large landscapes. Given SOD-wildfire interactions, further integration of disease- and fire-related management plans will be essential to minimizing impacts of these compounded disturbances.

In the Peruvian Amazon, white-sand forests are patchily distributed and restricted to a few localities in the North. Although recent studies have documented patterns of habitat specialization by plants in these unique forests, very few studies of the fauna of these habitats have been conducted. The species composition of the avifauna of the white-sand forests at six localities in the region was sampled by conducting transects and point counts. Surrounding habitats were also sampled to compare avifaunal communities and to determine the degree of restriction of bird species to white-sand habitats. Non-metric multidimensional scaling analysis showed that bird communities of white-sand forests were more similar to each other than they were to terra firme or flooded forest communities. Sites on either side of the Amazon-Mara~� barrier were the most similar within habitat type consistent with the hypothesis that these rivers represent a major biogeographic barrier. Twenty-six species, belonging to 13 families, were to some degree specialized to white-sand forests. This is the first comprehensive ornithological assessment carried out on these habitats in Peru. The high degree of habitat specialization found in these 26 bird species highlights the need for conservation and management measures that will protect white-sand forests.

Summary The first wildfires in sudden oak death-impacted forests occurred in 2008 in the Big Sur region of California, creating the rare opportunity to study the interaction between an invasive forest pathogen and a historically recurring disturbance. To determine whether and how the sudden oak death pathogen, Phytophthora ramorum, survived the wildfires, we completed intensive vegetation-based surveys in forest plots that were known to be infested before the wildfires. We then used 24 plot-based variables as predictors of P. ramorum recovery following the wildfires. The likelihood of recovering P. ramorum from burned plots was lower than in unburned plots both 1 and 2 yr following the fires. Post-fire recovery of P. ramorum in burned plots was positively correlated with the number of pre-fire symptomatic California bay laurel (Umbellularia californica), the key sporulating host for this pathogen, and negatively correlated with post-fire bay laurel mortality levels. Patchy burn patterns that left green, P. ramorum-infected bay laurel amidst the charred landscape may have allowed these trees to serve as inoculum reservoirs that could lead to the infection of newly sprouting vegetation, further highlighting the importance of bay laurel in the sudden oak death disease cycle.

Summary With growing incidence of bone disorders (such as osteoporosis), the demand for fast and reliable alternatives to existing screens based on mammalian bone is increasing. Cell culture-based models that currently are being used, have limited value in the study of cell-cell and cell-matrix interactions. The dermal bone of fish scales offers an unprecedented and attractive model to study bone physiology, both in vivo and in vitro. The elasmoid scale of teleost fishes is of particular interest as it could stand as a model for direct bone formation as found in mammals and it has many advantageous traits: scales carry both bone-forming and bone-degrading cell populations, attached on their natural substrate, such that interactions between cell populations remain intact. The scale contains a collagen type-I matrix impregnated with apatites. Scales come in the hundreds per fish, are easily harvested and can be exposed in vitro for direct study of bone regulatory compounds. Scales are transparent allowing real-time visualization of cellular activities on either side of the scale. Removed scales are replaced quickly and the regeneration process strongly resembles osteogenesis as seen in early bone development. These advantages warrant more fundamental research on scales as models in biomedical bone research. We here address the state-of-the-art on development, structure and physiology of fish scales.

Summary Collagen matrices, mineralized with calcium phosphates in a hydroxyapatite phase, are generally found in skeletal tissues. However, mechanisms of biomineral formation and regulation are still poorly understood. Elasmoid scales are part of the dermal skeleton and regenerate quickly when damaged or lost. This makes the scale a convenient target to study mineral compostion and formation. The X-ray diffractogram of the mineral layer of carp scales only show speaks corresponding with hydroxyapatite. Energy-dispersive X-ray spectroscopy (EDX) during scanning electron microscopy identifies the elements calcium and phosphorus, which are restricted to the external layer of the scale. In contrast with various other species, calcium phosphate crystals are not present in the elasmodine layer. The clean boundary between calcified and uncalcified matrix suggests that subsequental mineralization of the elasmodine layer is inhibited. Quantification of the calcium and phosphorus content allows definition of a calcium/phosphorus ratio, which is indicative ofthe crystalline phase of the minerals. Through accurate measurements of total calcium and phosphorus (by ICP-MS) in zebrafish scales, different ratios were found for newly formed (regenerating) scales compared to ontogenetic or completely regenerated scales. This shows that the mineral is not at first deposited as hydroxyapatite, but in a precursor phase. Although the nature of the phase remains to be established, this first analysis shows that cyprinids scales can easily be used to gain insights in the dynamics of biomineralization.

Summary 1. One of the earliest hypotheses to explain high tropical forest diversity proposes that species are differentially specialized in their germination or growth and survival to particular habitats. 2. I examined evidence for habitat associations in seedling density and demography in 9 years of seedling dynamics data from Yasuni National Park, Ecuador, a lowland rain forest where previous studies have demonstrated habitat preferences among adult trees. I included 136 species or morphospecies from multiple annual seedling cohorts with known age of recruitment. 3. Approximately 90% of the species examined demonstrated negative or positive associations with one or more topographic habitats in their recruitment, growth and/or mortality at some point in the study, and approximately 60% of species had significant associations in at least half of the census periods studied. The survival of newly recruited seedlings varied among seedlings in response to topographic gradients, indicating the potential for species to partition habitat niches at a young stage. 4. There was significant inter-annual variation in seedling habitat associations, indicating the characteristics of the topographic niche important to seedling performance change through time. This variability alone can contribute to the maintenance of species diversity through storage effects. While associations may also be weak or ephemeral, the seedling dynamics for many species supported the possibility that associations seen in adult populations develop through differential mortality across habitats as seedlings. 5. Synthesis. That species’ seedlings perform differently among topographic habitats and that these differences are detectable very early on in a plant’s life indicate the potential for the abiotic environment to mediate or exaggerate the roles of other mechanisms in influencing the composition of the understorey seedling assemblage.

Spatial and temporal variation in seedling dynamics was assessed using records of community-wide seedling demography collected with identical monitoring methods at four tropical lowland forests in Panama, Malaysia, Ecuador and French Guiana for periods of between 3 and 10 y. At each site, the fates of between 8617 and 391 777 seedlings were followed through annual censuses of the 370–1008 1-m2 seedling plots. Within-site spatial and inter-annual variation in density, recruitment, growth and mortality was compared with among-site variability using Bayesian hierarchical modelling to determine the generality of each site's patterns and potential for meaningful comparisons among sites. The Malaysian forest, which experiences community-wide masting, was the most variable in both seedling density and recruitment. However, density varied year-to-year at all sites (CVamong years at site = 8–43%), driven largely by high variability in recruitment rates (CV = 40–117%). At all sites, recruitment was more variable than mortality (CV = 5–64%) or growth (CV = 12–51%). Increases in mortality rates lagged 1 y behind large recruitment events. Within-site spatial variation and inter-annual differences were greater than differences among site averages in all rates, emphasizing the value of long-term comparative studies when generalizing how spatial and temporal variation drive patterns of recruitment in tropical forests.

The regulation of skin darkness in vertebrates is mediated by alpha-melanophore-stimulating-hormone (alphaMSH). For this action, alphaMSH binds to the melanocortin (MC)-1 receptor, a 7-transmembrane receptor located in melanophore cell membranes. The Mozambique tilapia, Oreochromis mossambicus, can change the hue of its body in response to a change in background, a process that may involve alphaMSH and the MC1R. Scale melanophores were isolated from tilapia that were acclimatised for 25 days to a black, control grey or white background and then tested for their sensitivity to des-, mono-, and di-acetylated alphaMSH. On all backgrounds, mono-acetylated alphaMSH was the dominant isoform present in pituitary homogenates. Mono-acetylated alphaMSH also had the highest potency to disperse melanosomes. Black background adapted fish showed the highest dispersing response to alphaMSH, independent of the isoform applied. We elucidated the nucleotide and amino acid sequence of the tilapia MC1R. We show that its expression in skin does not change when tilapia are acclimatised for 25 days to a black, grey or white background, while a clear change in hue is visible. This finding, combined with the absence of differential MC1R gene expression following background acclimation indicates that the increased sensitivity to alphaMSH is most likely a result of changes in the intracellular signalling system in melanophores of black background adapted fish, rather than up-regulation of the MC1R.

Contains fulltext : 33308.pdf (Publisher’s version ) (Closed access) Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.

Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.

Contains fulltext : 58054.pdf (Publisher’s version ) (Closed access) We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.

Isogenic carp Cyprinus carpio L. were acclimated to water temperatures of 15, 22 and 29 degrees C for at least 8 weeks. The acclimations consistently resulted in slightly, but significantly, different plasma osmolality, sodium, potassium and chloride concentrations between the groups studied. Plasma total and ionic calcium levels were unaffected, indicating successful adaptation. The apparent changes in set point for plasma ion levels are explained by altered sodium pump activity and hormonal control of branchial permeability to water and ions. It appears that in 15 degrees C-acclimated fish, a lower apparent Na(+)/K(+)-ATPase activity is compensated by strongly enhanced Na(+)/K(+)-ATPase expression (determined biochemically and immunohistochemically). In 29 degrees C-acclimated fish, the higher ambient temperature activates the enzyme. Arrhenius plots for branchial Na(+)/K(+)-ATPase preparations of the three groups of fish suggest the occurrence of different enzyme isoforms or protein (in)stability as explanations for differences in apparent enzyme activities, rather than temperature-dependent changes in membrane fluidity. As for hormonal control over permeability, prolactin mRNA expression (and anticipated production and release) is lower in fish kept at 29 degrees C, suggesting that control over branchial permeability to water and ions needs to be downregulated at higher temperatures. In so doing, enhanced sodium pump activity is balanced by a controlled passive ion loss to fine-tune plasma sodium levels. Basal plasma cortisol levels did not correlate positively with Na(+)/K(+)-ATPase expression, but doubling plasma cortisol levels in control fish by administering exogenous cortisol (for 7 days, using implanted minipumps and thus stress-free) enhanced Na(+)/K(+)-ATPase expression. This effect must be the result of a glucocorticoid action of the steroid: in fish, mineralocorticoid receptors have higher affinity for cortisol than glucocorticoid receptors. At a lower ambient temperature, branchial Na(+)/K(+)-ATPase expression is upregulated to counteract the temperature-inhibited activity of the sodium pump, perhaps via a mineralocorticoid receptor.

Tight interactions among different cell types contributing to bone formation are of key importance in the maintenance of bone homeostasis. Based on the high similarity in responses to (anti)osteogenic signals between zebrafish scales and mammalian bone, we developed and validated a model to screen large numbers of compounds using ex-vivo cultured scales of a sp7:luciferase transgenic zebrafish. This model combines the high predictive value of explant cultures with quick, sensitive, and quantifiable readout converging the effects via various pathways including WNT-signaling, to SP7/osterix promoter activity. Sp7 is pivotal in osteoblast differentiation and activity and its promoter activity provides an excellent surrogate for sp7 expression. Bmp-2a was shown to dose-dependently increase sp7-driven luciferase activity ex vivo. Next, we identified novel effects on bone for 51.7% of the compounds from a small library of WNT-signaling modulators, including a strong osteogenic effect for niclosamide. From all previously characterized compounds, the effect on bone was correctly predicted for 70% of compounds, resulting in a 7% false positive- and 21% false negative rate. The proposed sp7:luciferase zebrafish scale model is unique, powerful and efficient new tool to assess compounds with osteogenic effects, prior to further testing in rodents.

Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit alpha 1 of the Na+/K+-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

A melanotropin-like peptide (alpha-melanocyte-stimulating hormone or alpha-MSH) is suggested to be released into the circulatory system of cephalopods via the neurosecretory system of the vena cava or NSV, where neurosecretory vesicles contained within the axons of the NSV-neuropil on the inner surface of the vena cava lie in close contact with the venous circulation. Radioimmunoassay of blood plasma samples taken from the cephalic vein of anaesthetised cuttlefish, Sepia officinalis showed that immunoreactive alpha-MSH (ir alpha-MSH) was detectable within the cuttlefish circulatory system. The validity of the assay for determination of cuttlefish ir alpha-MSH was determined by parallelism of the alpha-MSH standard curve against serially diluted cuttlefish plasma samples. Plasma samples taken during a natural day-night-day illumination cycle showed a significant elevation in ir alpha-MSH concentration to 1.44 +/- 0.26 ng ml(-1) during the middle of the dark phase compared to concentrations of 0.48 +/- 0.13 and 0.35 +/- 0.10 ng ml(-1) in the middle of the light phases of the illumination cycle. So far, indirect evidence suggests Sepia officinalis may modulate chromatophore activity, body patterning, and behaviour via neuroendocrine release and circulating titres of this proopiomelanocortin-derived peptide.

Although there is growing recognition of the importance of small mammals in savanna ecosystems of east Africa, the ecology and behavior of these organisms are not well known. We conducted studies on the dietary preferences of one rodent, the pouched mouse (Saccostomus mearnsi), which is the dominant small mammal in a widespread habitat in central Kenya. To establish the food preferences of this species, we: (1) conducted cafeteria trials in the laboratory whereby individual mice were offered seeds and a selection of herbaceous vegetation from the habitat; (2) surveyed the habitat for piles of vegetation clipped by these mice and identified these clippings to species; and (3) estimated the abundance of plant species in the habitat to determine if the mice were clipping species in proportion to their availability. Our results indicated that S. mearnsi prefers green vegetation to seeds, at least during wet seasons, and that it has a strong preference for forbs. In cafeteria trials, S. mearnsi chose ...

Objectives: Evaluation of an aggressive policy for the treatment of phrenic nerve palsy (PNP), following cardiac operations, with emphasis on early diaphragmatic plication. Attention was given to the incidence and predisposing factors for PNP and the potential for recovery following plication. Methods: From 1 June 1991 to 1 January 1996 we prospectively screened patients for PNP following cardiac surgery. The diagnosis was suspected if difficulty was experienced in weaning the child from the ventilator. If abnormal elevation of the hemidiaphragm was present diaphragmatic plication was performed. Echocardiography was used to assess subsequent return of diaphragmatic function. Results: Seventeen children (nine boys, eight girls), out of 867 (1.9%) children younger than 16 years of age, undergoing cardiac operations were found to have PNP. The mean age was 66 days (range 1‐17 months) with 16 patients below 1 year out of a total of 285 patients (incidence 5.6%) and one patient 17 months old. The incidence following open procedures was 11/190, following closed procedures 2/95 and following reoperation 4/83. PNP was diagnosed from 2 to 44 days (mean 14 days) following surgery. It was present on the right side in seven cases, the left in nine and was bilateral in one patient. Two patients were extubated at the time of diagnosis, one patient could be extubated shortly thereafter. Fourteen children underwent diaphragmatic plication, at a median 5 days post diagnosis. Extubation was possible 1‐60 days (mean 4 days) after plication. Mean follow-up was 19 ∠ 5 months. Subsequent recovery of diaphragmatic movement was documented in seven (41%) children. Time to recovery following plication was 16 months, without plication 38 months. Conclusion: Prospective screening for PNP revealed an incidence in children younger than 1 year of 6%. Early plication substantially reduces the duration of ventilation, with its associated reduced morbidity and ICU stay. © 1998 Elsevier Science B.V. All rights reserved

Corticotropin-releasing factor-binding protein (CRF-BP) is considered a key determinant for CRF receptor (CRF-R) activation by CRF and several related peptides. Earlier studies have shown that the CRF system is highly conserved in gene structures throughout evolution, yet little is known about the evolutionary conservation of its biological functions. Therefore, we address the functional properties of CRF-BP and CRF-Rs in a teleost fish (common carp; Cyprinus carpio L.). We report the finding of two similar, yet distinct, genes for both CRF-R1 and CRF-R2 in this species. The four receptors are differentially responsive to CRF, urotensin-I (UI), sauvagine, and urocortin-2 (Ucn-2) and -3 (Ucn-3) as shown by luciferase assays. In vitro, carp CRF-BP inhibits CRF- and UI-mediated activation of the newfound CRF-Rs, but its potency to do so varies between receptor and peptide ligand. This is the first paper to establish the functionality and physiological interplay between CRF-BP, CRF-Rs and CRF-family peptides in a teleostean species.

Summary Calcium is of vital importance in vertebrates and plasma levels are tightly regulated. Terrestrial vertebrates depend for their calcium uptake exclusively on the diet, while fish have an essentially infinite directly accessible source of calcium in the surrounding water. The need to store calcium seems thus less evident in fish. It was the goal of the present study to investigate the possible role of the scale compartment of zebrafish (Danio rerio) as an accessible pool for calcium and phosphorus. We restricted the calcium availability to zebrafish both in water and diet. Following 12 days restriction, we found that scalar contents of calcium and phosphorus had declined, while magnesium was unaffected, indicating resorption of calcium hydroxyapatite. Osteoclast activity on scales of calcium-restricted fish increased, as we conclude from enlarged demineralized areas on scales, increased TRAcP activity staining, and higher ctsk gene expression. Osteoblasts respond to calcium limitation by increasing expression of col1a2 and alpl. In the presence of water and dietary calcium, removal of scales from one flank of the fish does not affect scalar mineral contents, scale demineralization and osteoclastic TRAcP activity; expression of sp7, alpl and ctsk are up-regulated. Apparently, a high demand for calcium in zebrafish is preferentially complemented with exogenous calcium, while calcium is recruited from the scales when environmental calcium is limited. We conclude that the scales represent an important deposit of available calcium and phosphorus from which these minerals can be recruited in periods of high demand. This finding contributes to a niche for scales as model in bone research.

Item does not contain fulltext We demonstrate that glucocorticoids induce an osteoporotic phenotype in regenerating scales of zebrafish. Exposure to prednisolone results in altered mineral content, enhanced matrix breakdown, and an osteoporotic gene-expression profile in osteoblasts and osteoclasts. This highlights that the zebrafish scale provides a powerful tool for preclinical osteoporosis research. INTRODUCTION: This study aims to evaluate whether glucocorticoid (prednisolone) treatment of zebrafish induces an osteoporotic phenotype in regenerating scales. Scales, a readily accessible dermal bone tissue, may provide a tool to study direct osteogenesis and its disturbance by glucocorticoids. METHODS: In adult zebrafish, treated with 25 muM prednisolone phosphate via the water, scales were removed and allowed to regenerate. During regeneration scale morphology and the molar calcium/phosphorus ratio in scales were assessed and osteoblast and osteoclast activities were monitored by time profiling of cell-specific genes; mineralization was visualized by Von Kossa staining, osteoclast activity by tartrate-resistant acid phosphatase histochemistry. RESULTS: Prednisolone (compared to controls) enhances osteoclast activity and matrix resorption and slows down the build up of the calcium/phosphorus molar ratio indicative of altered crystal maturation. Prednisolone treatment further impedes regeneration through a shift in the time profiles of osteoblast and osteoclast genes that commensurates with an osteoporosis-like imbalance in bone formation. CONCLUSIONS: A glucocorticoid-induced osteoporosis phenotype as seen in mammals was induced in regenerating scalar bone of zebrafish treated with prednisolone. An unsurpassed convenience and low cost then make the zebrafish scale a superior model for preclinical studies in osteoporosis research. 01 februari 2014

An under-examined component of global change is the alteration of disturbance regimes due to warming climates, continued species invasions, and accelerated land-use change. These drivers of global change are themselves novel ecosystem disturbances that may interact with historically occurring disturbances in complex ways. Here we use the natural experiment presented by wildfires in redwood forests impacted by an emerging infectious disease to demonstrate unexpected synergies of novel disturbance interactions. The dominant tree, coast redwood (fire resistant without negative disease impacts), experienced unexpected synergistic increases in mortality when fire and disease co-occurred. The increased mortality risk, more than fourfold at the peak of the effect, was not predictable from impacts of either disturbance alone. Changes in fire behavior associated with changes to forest fuels that occurred through disease progression overwhelmed redwood's usual resilience to wildfire. Our results demonstrate the potential for interacting disturbances to initiate novel successional trajectories and compromise ecosystem resilience.

Herbivores are often implicated in the generation of the extraordinarily diverse tropical flora. One hypothesis linking enemies to plant diversification posits that the evolution of novel defenses allows plants to escape their enemies and expand their ranges. When range expansion involves entering a new habitat type, this could accelerate defense evolution if habitats contain different assemblages of herbivores and/or divergent resource availabilities that affect plant defense allocation. We evaluated this hypothesis by investigating two sister habitat specialist ecotypes of Protium subserratum (Burseraceae), a common Amazonian tree that occurs in white-sand and terra firme forests. We collected insect herbivores feeding on the plants, assessed whether growth differences between habitats were genetically based using a reciprocal transplant experiment, and sampled multiple populations of both lineages for defense chemistry. Protium subserratum plants were attacked mainly by chrysomelid beetles and cicadellid hemipterans. Assemblages of insect herbivores were dissimilar between populations of ecotypes from different habitats, as well as from the same habitat 100 km distant. Populations from terra firme habitats grew significantly faster than white-sand populations; they were taller, produced more leaf area, and had more chlorophyll. White-sand populations expressed more dry mass of secondary compounds and accumulated more flavone glycosides and oxidized terpenes, whereas terra firme populations produced a coumaroylquinic acid that was absent from white-sand populations. We interpret these results as strong evidence that herbivores and resource availability select for divergent types and amounts of defense investment in white-sand and terra firme lineages of Protium subserratum, which may contribute to habitat-mediated speciation in these trees.

Proper functioning of the endocrine stress axis requires communication between the stress axis and other regulatory mechanisms. We here describe an intimate interplay between the stress axis and recombinant human leptin (rhLeptin) in a teleostean fish, the common carp Cyprinus carpio. Restraint stress (by netting up to 96h) increased plasma cortisol but did not affect hepatic leptin expression. Perifusion of pituitary glands or head kidneys with rhLeptin revealed direct effects of rhLeptin on both tissues. RhLeptin suppresses basal and CRF-induced ACTH-secretion in a rapid and concentration-dependent manner. The rhLeptin effect persisted for over an hour after administration had been terminated. RhLeptin decreases basal interrenal cortisol secretion in vitro, and by doing so attenuates ACTH-stimulated cortisol production; rhLeptin does not affect interrenal ACTH-sensitivity. Our findings show that the endocrine stress axis activity and leptin are inseparably linked in a teleostean fish, a notion relevant to further our insights in the evolution of leptin physiology in vertebrates.

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

Transcriptional regulation in eukaryotes is thought to occur through interactions between specific transcription factors and the general transcription machinery. We show that the regulatory protein FosB, but not FosB/SF or Fra-1, specifically and stably associates with the TATA box binding protein (TBP) and the multiprotein complex TFIID. The binding to TBP is specified by the last 55 C-terminal amino acids of FosB, requiring a small amino acid sequence, termed the 'TBP binding motif' (TBM). Deletion of the TBM affects transcriptional activity slightly, but it is adjacent to a proline-rich sequence which constitutes the major transactivation domain. However, both regions are required for the transformation of Rat-1A cells by FosB. Transfection experiments demonstrate that inhibition of transactivation due to excess levels of Gal4-FosB (squelching) can be partially relieved by the co-expression of TBP, which establishes that TFIID is a functional target of FosB. Since TBP binding is not exhibited by FosB/SF or Fra-1, we suggest that the activity mediated by the TBP interaction is one differentiating characteristic that distinguishes the FosB functions from those of FosB/SF and Fra-1.

The precursor protein proopiomelanocortin (POMC) gives rise to a variety of biologically active peptides through cell-specific posttranslational processing. Two transcripts of pomc were found in the flatfish Solea senegalensis (ssePOMC-A and ssePOMC-B), that most likely represent subfunctionalized paralogues: ssePOMC-A lacks the N-terminal cleavage site for β-MSH, whereas ssePOMC-B cannot yield ACTH and completely lacks the opioid consensus sequence in the β-END region. An analysis of nucleotide substitution rates shows that the POMC-derived peptides possess well-conserved regions under purifying selection, except the β-END derived from POMC-B, which has undergone positive selection. The calculated Ks values for ssePOMC-A versus ssePOMC-B and zebrafish POMCα versus zebrafish POMCβ are 0.40 and 0.72, respectively, indicating that the zebrafish POMC paralogues started to evolve almost twice as early in evolution, and that the Solea POMC paralogues arose independently from the whole genome duplication event that gave rise to the zebrafish paralogues. This makes ssePOMC-B the first identified POMCα orthologue that lacks the opioid consensus. Furthermore, pomc-a expression is down-regulated in chronic stressed S. senegalensis juveniles, whereas pomc-b expression levels remain unaffected, indicating different physiological roles for both POMC paralogues. The distribution of functional POMC-derived peptide hormones over two pomc genes in S. senegalensis suggests subfunctionalization of the paralogues, a relevant notion when studying POMC function in endocrine responses., This work was partially funded by grants AGL2007-61211/ACU (Ministerio Educación y Ciencia of Spain) and Proyecto de Excelencia PO7-RNM-02843 (Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía) to J.M.M. The authors are grateful to “Planta de Cultivos Marinos” (CASEM, Puerto Real, Spain) for the donation of S. senegalensis. Y.W. is funded by Ministerio de Educación y Ciencia (Spain) through the program “Formación de Profesorado Universitario” (Ref: AP2006-03932). E.V. is funded by the SmartMix Program of the Netherlands Ministry of Economic Affairs and the Ministry of Education, Culture and Science.

Calcium is an essential ion serving a multitude of physiological roles. Aside from its role as a second messenger, it is an essential component of the vertebrate bone matrix. Efficient uptake and storage of calcium are therefore indispensable for all vertebrates. Transient receptor potential family, vanilloid type (TRPV)5 and TRPV6 channels are known players in transcellular calcium uptake, but the exact contribution of this pathway is unclear. We used forward genetic screening in zebrafish (Danio rerio) to identify genes essential in bone formation and identified a lethal zebrafish mutant (matt-und-schlapp) with severe defects in bone formation, including lack of ossification of the vertebral column and craniofacial structures. Mutant embryos show a 68% reduction in calcium content, and systemic calcium homeostasis is disturbed when compared with siblings. The phenotype can be partially rescued by increasing ambient calcium levels to 25 mM. We identified the mutation as a loss-of-function mutation in the single orthologue of TRPV5 and 6, trpv5/6. Expression in HEK293 cells showed that Trpv5/6 is a calcium-selective channel capable of inward calcium transport at physiological concentrations whereas the mutant channel is not. Taken together, this study provides both genetic and functional evidence that transcellular epithelial calcium uptake is vital to sustain life and enable bone formation. [KEYWORDS: Animals, Bone Development/genetics/ physiology, Calcium/ metabolism, Embryo, Nonmammalian/metabolism, Epithelium/ embryology/ metabolism, Gene Expression Regulation, Developmental/physiology, HEK293 Cells, Humans, Molecular Sequence Data, Mutation, TRPV Cation Channels/genetics/ metabolism, Zebrafish/ embryology, Zebrafish Proteins/genetics/ metabolism]

Negative density-dependent mortality can promote species coexistence through a spacing mechanism that prevents species from becoming too locally abundant. Negative density-dependent seedling mortality can be caused by interactions among seedlings or between seedlings and neighboring adults if the density of neighbors affects the strength of competition or facilitates the attack of natural enemies. We investigated the effects of seedling and adult neighborhoods on the survival of newly recruited seedlings for multiple cohorts of known age from 163 species in Yasuni National Park, Ecuador, an ever-wet, hyper-diverse lowland Amazonian rain forest. At local scales, we found a strong negative impact on first-year survival of conspecific seedling densities and adult abundance in multiple neighborhood sizes and a beneficial effect of a local tree neighborhood that is distantly related to the focal seedling. Once seedlings have survived their first year, they also benefit from a more phylogenetically dispersed seedling neighborhood. Across species, we did not find evidence that rare species have an advantage relative to more common species, or a community compensatory trend. These results suggest that the local biotic neighborhood is a strong influence on early seedling survival for species that range widely in their abundance and life history. These patterns in seedling survival demonstrate the role of density-dependent seedling dynamics in promoting and maintaining diversity in understory seedling assemblages. The assemblage-wide impacts of species abundance distributions may multiply with repeated cycles of recruitment and density-dependent seedling mortality and impact forest diversity or the abundance of individual species over longer time scales.