The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.The conventional model of intestinal epithelial architecture describes a unidirectional tissue organizational hierarchy with stem cells situated at the crypt base and daughter cells proliferating and terminally differentiating as they progress along the vertical  axis. In this model, the fate of a cell that has left the niche is determined and its lifespan limited. Evidence is accumulating to suggest that stem cell control and daughter cell fate determination is not solely an intrinsic, cell autonomous property but is heavily influenced by the microenvironment including paracrine, mesenchymal, and endogenous epithelial morphogen gradients. Recent research suggests that in intestinal homeostasis, stem cells transit reversibly between states of variable competence in the niche. Furthermore, selective pressures that disrupt the homeostatic balance, such as intestinal inflammation or morphogen dysregulation, can cause committed progenitor cells and even some differentiated cells to regain stem cell properties. Importantly, it has been recently shown that this disruption of cell fate determination can lead to somatic mutation and neoplastic transformation of cells situated outside the crypt base stem cell niche. This paper reviews the exciting developments in the study of stem cell dynamics in homeostasis, intestinal regeneration, and carcinogenesis, and explores the implications for human disease and cancer therapies. © 2015 Authors.  The intestinal epithelium is a paradigm for the systems governing cellular renewal in the epithelial tissues that line our body. The gut mucosa has evolved to selectively absorb nutrients and water whilst protecting the body from the toxic contents of the gut lumen. This protection is afforded by the continuous renewal of the epithelium along the vertical  axis of the intestine, allowing migrating, differentiated epithelial cells that have been exposed to genotoxic luminal contents to apoptose and be shed into the gut lumen. Intestinal epithelial turnover depends on a self‐renewing stem cell population with rapid expansion of stem cell progeny followed by differentiation of the daughter cell population, all within the 5–7 days that it takes for a cell to migrate from the crypt base to the luminal surface. The hierarchical and stereotypical architecture of the intestinal epithelium makes it an attractive model for the study of tissue‐specific stem cells, alongside the mechanisms that control cell fate determination – processes that are fundamental for homeostasis and frequently deranged in carcinogenesis.Experimental approaches to study these mechanisms are reliant on the ability to accurately identify stem cells and then trace the fate of their progeny. Meticulous, pioneering work in the 1970s used radiation sensitivity and surrogate markers to identify two distinct populations of cells with stem‐like properties – crypt base columnar (CBC) cells This review will explore some of the recent advances in our understanding of the control of the intestinal stem cell and daughter cell fate determination in intestinal homeostasis and assess the implications for colorectal carcinogenesis.DCLK1 (doublecortin‐like kinase 1) , which is restricted to the crypt base columnar cells Lgr5‐positive transgenically labelled crypt base columnar cells. Lgr5 has subsequently been validated as a bona fide stem cell marker in the small intestine, colon By focusing on Wnt signalling as the main pathway controlling stem cell function, the Clevers group established a panel of Wnt target genes and used Lgr5 cells which divide about once a day, the cells in the +4 position of the crypt, also defined by the property of label retention, are quiescent and slowly cycling. Several cell markers with expression patterns that overlie this position have been described Bmi1 Cre recombinase mouse, Sangiorgi et al demonstrated lineage tracing from cells at the +4 position Bmi1 is in fact expressed broadly throughout the crypt, diminishing this as a specific stem cell marker et al focused on cells with functional label retention properties and in a series of elegant experiments, demonstrated that these cells are committed secretory cell precursors that retain the capability of returning to stem cell function in the event of intestinal damage and regeneration Tert, Hopx, Lrig1, Sox9, Ascl2, Olfm4, Prom1, and Rnf43In contrast to the Regulation of normal intestinal stem cells occurs within a discrete microenvironment confined to the crypt base, known as the stem cell niche. This consists of epithelial and mesenchymal cells and extracellular substrates which favour the existence of a stem cell in its undifferentiated state. It provides an optimal microenvironment for the production of differentiated progeny by the paracrine secretion of growth factors, cytokines, and morphogens (soluble molecules produced by a restricted region of a tissue that form an activity gradient away from its source). The phenotypic response of a cell is determined by its position within these concentration gradients. Key constituents of the niche include Paneth cells and pericryptal fibroblasts, which provide critical factors for the survival of crypt base stem cells such as Wnt3a, epidermal growth factor (EGF), and bone morphogenetic protein (BMP) antagonists Adult stem cell and daughter cell fate determination is controlled by the same signalling pathways that regulate embryonic stem cell function during development. In the adult, these pathways are stringently controlled with complex interactions used to restrict pathway activity and response to the appropriate cell compartment. Mesenchymal and epithelial‐derived pathways result in polarized gradients that regulate stemness, cell proliferation, differentiation, and apoptosis as cells progress along the intestinal vertical axis. Important pathways include Wnt, BMP, Hedgehog, and Notch signalling Figure .Drosophila to man. Beta‐catenin is the key link in the pathway. Usually anchored to the basement membrane, intracellular β‐catenin is minimized by phosphorylation and ubiquitin‐proteasome degradation of its complex with adenomatous polyposis coli (APC). Binding of Wnt to Frizzled receptors inhibits degradation of β‐catenin, so increasing its cytosolic level. This results in transcription of target genes driving proliferation. The non‐canonical Wnt pathway, which does not involve β‐catenin, is also essential for epithelial proliferation. In the intestine, Wnt signalling is mainly restricted to the stem and proliferative zones in the bottom third of the crypt, where it is involved in maintenance of stem cell function and driving transit amplifying cell proliferation.This highly conserved signalling pathway involved in cell fate specification is expressed in species ranging from Gremlin1, 2, and, to a lesser extent, Noggin.BMP signalling is part of the TGFβ (transforming growth factor β) superfamily and phosphorylates Smad proteins for signal transduction. BMP signalling has a pivotal role in intestinal development and is required for the control of intestinal stem cell replication. It is also needed for terminal differentiation of mature intestinal cells Hedgehog controls tissue polarity. Indian hedgehog is the main Hedgehog protein expressed in the intestine and is secreted in a paracrine manner by differentiated epithelial cells to act on mesenchymal cells. It maintains homeostasis of mesenchymal cells and regulates epithelial cell proliferation through negative feedback to proliferating crypt base columnar cells by increasing BMP signalling Math1 genevia the transcriptional repressor Hes1. Lateral inhibition in this cell‐to‐cell signalling pathway means that adjacent cells are driven towards different fates, resulting in a ‘salt and pepper’ distribution of progenitor cells committed to enterocyte and secretory lineages Notch interacts with Delta and Jagged ligands and signals through cell–cell contact to regulate post‐mitotic differentiation into the different cell lineages. It thus regulates cell fate decision between absorptive and secretory cell types. Notch signalling is also involved in the control of stem cells and transit amplifying cell division et alin vivo. Quantitative analysis of individual clonal lineages showed that ‘central cells’ at the crypt base, optimally located near niche‐derived signals, had a survival advantage over ‘border cells’ in the upper part of the niche. Compared with their centrally located counterparts, border cells were more likely to be displaced into the transit‐amplifying compartment, lose stem cell properties, and progress along the crypt–villus escalator. This indicates that there is a spectrum of stem cell function even within cells expressing the Lgr5 marker and that these cells transit reversibly between states of variable competence influenced by proximity to niche‐derived signals. Somatic events such as Apc and Kras mutations that bestow a selective advantage can bias a stem cell towards clonal expansion within the niche, although Vermeulen et alTp53 inactivation only resulted in a selective advantage and resultant niche succession in an inflammatory microenvironment, which highlights the critical interplay between genotype and environmental context in disrupted homeostasis and colorectal carcinogenesis.It is increasingly recognized that stem cell function is not an intrinsic, cell autonomous property and that stem cell fate is not fixed, even within the confines of the stem cell niche. Elegant work by the Winton and Clevers groups has shown that murine intestinal stem cells form an equipotent population that undergoes stochastic clone extinction, compensated by expansion and replacement by a neighbouring cell. This random enlargement and contraction of clones, known as neutral drift dynamics, can lead to stochastic clonal extinction or niche succession, where one clone expands to fill the entire niche The intestinal epithelium is the first line of defence in a hostile environment and is subject to frequent chemical, microbiological, and immunological mediated mucosal damage. Many of the evolutionarily conserved pathways that control cell fate determination in the intestine are also involved in the physiological response to damage and intestinal regeneration. Regulated disruption of these epithelial, stromal, and immunological morphogen gradients with resultant change in epithelial stem cell function is a key feature of the response to damage and underpins the regenerative and reparative capacity of the intestinal epithelium. Lineage tracing studies using different stem cell markers have defined a number of cell populations enriched for stem cell potential/activity within the base of the crypt and dynamic studies using the most established markers have shown considerable variability in stem cell competence even during intestinal homeostasis. Recent work using specific killing of a single marker‐defined cell population or more widespread damage has helped to assess stem and daughter cell dynamic changes when homeostasis is disrupted.Lgr5+ cells in mice caused no phenotypic change because of the compensatory effects of an expansion in the Bmi1+ cell population Lgr5‐negative, notch ligand Delta like 1‐expressing secretory progenitors resulted in full crypt–villus lineage tracing following intestinal radiation et al showed that intestinal architecture can regenerate if either the stem cell or the progenitor zone remains intact following injury; however, if both cell populations are damaged/ablated simultaneously, the intestinal architecture is irreversibly perturbed Within the stem cell niche, genetic ablation of a specific stem cell causes a reserve pool to step up to prevent an overall phenotypic change et al used a doublecortin‐like kinase1 transgenic mouse to label and trace the fate of mature tuft cells in the murine colon. Surprisingly, they showed that these quiescent post‐mitotic, differentiated cells survived outside of the crypt base for many months, resisting the stream of enterocytes and goblet cells passing along the crypt–villus escalator. Rare lineage tracing events in the stomach and intestine were seen from transgenically labelled tuft cells and genetic ablation of these cells impaired the ability of the intestine to respond to radiation‐ or colitis‐induced damage These findings oppose our dogmatic understanding of a strict unidirectional tissue organizational hierarchy in the intestine and raise questions over the limits of intestinal cell plasticity. If progenitor cells are capable of dedifferentiation and return to stem cell function following injury, is the fate of fully differentiated cells fixed? Westphalen Thus, it appears that regulated, temporary disruption of morphogen gradients in a damaged mucosa is essential to alter stem cell dynamics, dedifferentiate progenitor and even some differentiated cells, and promote epithelial cell migration and proliferation to effect epithelial restitution Lgr5+ crypt base columnar cells, which was a stark contrast to the small non‐progressive phenotype provoked by introducing the same mutations into non‐stem cells The cellular origin of colorectal neoplasia has been controversial, centred on the mechanism of clonal expansion in human colorectal precursor lesions. The ‘top down’ model was based on the observation that some early adenomatous lesions develop on the luminal surface without any obvious contact to the crypt base and proposed an intercrypt location for the stem cell niche. The ‘bottom up’ model was founded on the more conventional crypt base stem cell compartment and an assumed unidirectional tissue organizational hierarchy. This model proposed that dysplasia arose in the stem cells at the crypt base, with subsequent spread of dysplastic progeny along the length of the crypt et al used mouse models to demonstrate that activated nuclear factor‐kappa B‐induced mucosal inflammation, in combination with constitutive epithelial Wnt signalling transgenically targeted to the non‐stem cell population, could induce dedifferentiation of Lgr5‐negative cells situated outside of the crypt base stem cell niche. Critically, they also showed that it was these dedifferentiated villus cells that gave rise to the tumours that rapidly arose in these animals Apc mutations to long‐lived, quiescent tuft cells had no phenotypic effect in a normal environmental context, but gave rise to aggressive, poorly differentiated carcinoma when intestinal inflammation was induced with dextran sodium sulphate, highlighting the dependence of both cell‐intrinsic (genetic) and cell‐extrinsic  factors in promoting carcinogenic initiation in this situation GREM1, which results in a compartmental expression switch – from a restricted mesenchymal gradient to ectopic gene expression throughout the epithelium Grem1 expression developed an HMPS phenotype with mixed morphology polyps arising from ectopic crypts. We demonstrated that this disruption of polarized homeostatic BMP signalling gradients led to the expansion of an Lgr5‐negative progenitor cell population into ectopic crypts. These ECF progenitor cells actively proliferate and accumulate somatic mutations, eventually resulting in intestinal neoplasia arising outside the crypt base stem cell niche  and environment (subdivided into cell‐intrinsic and cell‐extrinsic environment) Figure . The relThe implications of this conceptual shift can be interpreted in light of the recent evidence demonstrating the molecular heterogeneity of colorectal cancer. Several recent publications have sought to classify and subtype colorectal cancer based on (epi)genetic mutation and gene expression pattern The notion of a unidirectional tissue organizational hierarchy in the intestine has provided a useful conceptual framework to support our understanding of the location and function of intestinal stem cells, the determination of daughter cell fate, and the cell of origin of colorectal cancer. Recently, the ability to selectively manipulate the intestinal microenvironment and the increasing sophistication of the transgenic animal tools used to assess stem cell dynamics have shown that this model requires re‐evaluation. Stem cells are defined functionally by the characteristics of self‐renewal and multi‐lineage potential, not as individual cells determined by the expression of one or more markers. The work in this review highlights that the stem cell pool should be thought of as a functional collective, comprising a dynamic and heterogeneous population of cells with variable stem cell competence imposed by position within the niche and microenvironmental morphogen context.Colorectal cancer remains a paradigm for the cancer stem cell model, which proposes that tumours are maintained by a small population of tumourigenic, self‐renewing cancer stem cells that are at the apex of a cellular hierarchy, not dissimilar to that seen in normal tissue. The bulk of the tumour is thus made up of non‐clonogenic daughter cells that have initiated organ‐specific differentiation programmes and have limited tumourigenic and metastatic potential. There is evidence to support this assertion: single colorectal cancer cells are capable of multi‐lineage differentiation et al identified myofibroblast‐derived hepatocyte growth factor (HGF) as an activator of β‐catenin‐dependent transcription and resultant epithelial cancer stem cell clonogenicity osteopontin and SDF1 to impose a cancer stem cell phenotype on CRC progenitor cells IL‐22, which acts through activation of STAT3 signalling and DOT1L methyltransferase to influence cancer stem cell stemness. Neutralization of IL‐22 signalling can attenuate and even reverse an inflammation‐driven mouse model of colorectal cancer In contrast to the exquisitely controlled and balanced morphogen signalling seen in intestinal homeostasis, the cancer stem cell microenvironment is a disorganized and chaotic signalling mix, originating from cancer‐associated fibroblasts, infiltrating immunocytes, and endothelial cells. Working with primary CRC cultures, Vermeulen Jagged1GREM1 expression is associated with colorectal cancer subtypes that respond poorly to current chemotherapeutic regimes and have a dismal outcome GREM1 in imposing a stem cell phenotype on cells outside the crypt base, it seems plausible that the poor treatment response may be secondary to an effect of GREM1 on cancer stem cell plasticity. The identification of disrupted signalling pathways that influence cancer stem cell plasticity could open up therapeutic targets that aim to redress the signalling balance in tumours. Resetting the morphogen balance in tumours could reduce the speed or effectiveness of CSC dynamic change in response to chemotherapeutic selective pressures, allowing current drugs to kill cancer stem cells more effectively.Even tumour‐associated endothelial cells have recently been shown to have an effect on the cancer stem cell phenotype by paracrine secretion of the Notch ligand As experimental techniques improve, our understanding of the position, fate, and role of intestinal stem cells in homeostasis and carcinogenesis is evolving. The intestinal stem cell pool is a dynamic population of cells whose fate is heavily influenced by the microenvironmental signals received from surrounding supporting tissue. The epithelial response to disruption of these signals is a critical feature of the intestine's ability to respond to injury, yet signalling pathway dysregulation is also a key feature in the initiation of cancer. Teasing apart how to manipulate disrupted signalling in tumours without affecting the same pathway's role in tissue homeostasis is the critical challenge for future drug design. To understand and then target the factors that discriminate the neoplastic from the normal stem cell niche will be the next step.SB and SL wrote the article. HD, SI, TS, and DW reviewed and edited the manuscript.


Floods are a natural hazard that affect communities worldwide, but to date the vast majority of flood hazard research and mapping has been undertaken by wealthy developed nations. As populations and economies have grown across the developing world, so too has demand from governments, businesses, and NGOs for modeled flood hazard data in these data‐scarce regions. We identify six key challenges faced when developing a flood hazard model that can be applied globally and present a framework methodology that leverages recent cross‐disciplinary advances to tackle each challenge. The model produces return period flood hazard maps at ∼90 m resolution for the whole terrestrial land surface between 56°S and 60°N, and results are validated against high‐resolution government flood hazard data sets from the UK and Canada. The global model is shown to capture between two thirds and three quarters of the area determined to be at risk in the benchmark data without generating excessive false positive predictions. When aggregated to ∼1 km, mean absolute error in flooded fraction falls to ∼5%. The full complexity global model contains an automatically parameterized subgrid channel network, and comparison to both a simplified 2‐D only variant and an independently developed pan‐European model shows the explicit inclusion of channels to be a critical contributor to improved model performance. While careful processing of existing global terrain data sets enables reasonable model performance in urban areas, adoption of forthcoming next‐generation global terrain data sets will offer the best prospect for a step‐change improvement in model performance. High‐resolution flood hazard model that employs globally available data setsQuantitative assessment of model performance relative to benchmark local dataPerformance adequate for certain real world applications in data‐scarce regions Historically, our understanding of global flood hazard has been limited by the poor monitoring of river networks in many regions [Ngo‐Duc et al., Pappenberger et al., Yamazaki et al. [Winsemius et al., Gouldby et al., Neal et al., Sampson et al., Ward et al., Winsemius et al., Flood hazard maps are designed to indicate the probability of flooding over space and serve as a critical decision‐making tool for a range of end users including building/infrastructure developers and disaster response planners. However, such maps do not exist for much of the developing world due to the extremely high data and computational requirements of the engineering hydraulic models that have traditionally been used in their production. Existing research toward more tractable simplified global scale models of surface water flows has typically been limited to hydrological routing schemes that are driven by regional or global climate models. One of the more sophisticated schemes of this type involved linking the Total Runoff Integrating Pathways 2 (TRIP2) global river network [i et al.  presenteda Paz et al., de Paiva et al., Biancamaria et al., Neal et al., Jung et al., Schumann et al., de Paiva et al., Biancamaria et al., da Paz et al., Neal et al., Schumann et al., Feyen et al. [Alfieri et al., Over recent years, the large gap that exists between simplified large‐scale approaches and detailed reach scale hydraulic models has begun to close as the result of significant research advances and increasing computational and data resources. A number of detailed hydraulic models  have been constructed for large river reaches in data sparse regions including the Amazon [n et al.  producedIn this paper, we propose a methodology that enables the construction of a global flood model for flood hazard assessment at ∼90 m spatial resolution where local detailed data and observations are not available. Models of this type are already being developed by the private sector in the insurance market for underwriting purposes in developing regions, but the detailed methods underlying such commercial models are not always clear and public domain validation exercises are limited. Validation of these methods is crucial to ensure that they can be applied appropriately through a proper understanding of their strengths and limitations. We argue there are six key challenges that need to be overcome to allow the development of such models, and that the state of science has advanced to the point whereby each of these challenges can now be tackled. The resulting global model is sufficiently resolved to enable direct benchmarking against high‐resolution local and national flood hazard data sets—to our knowledge the first time such an exercise has been undertaken for a model of this type. The framework and methods presented here should be considered a starting point, and we identify a number of areas where significant improvement may be achieved through targeted future research.Global terrain data (green).Extreme flow generation (blue).Global river network and geometry (yellow).Flood defenses (purple).Computational hydraulic engine (orange).Automation framework (red).Below we have identified six key challenges that need to be solved to enable a global flood model to be built. The bracketed colors in the list below associate each challenge with a region of the methodological flowchart Figure .Global For each challenge, we review relevant recent advances in the literature before presenting the approach we have adopted. It should be noted at this point that the above is not an exhaustive list of challenges for this topic, and others not yet handled by this framework are introduced as limitations in the discussion section.11.1Farr et al., Rabus et al., Hirt et al., Jing et al., Rexer and Hirt, Abrams, Fujisada et al., Jing et al., Rexer and Hirt, Kervyn, Reuter et al., Reuter et al. [Lehner et al., Jarvis et al., Kobrick, Gallant, Gallant and Read, Hutchinson et al., Stevenson et al., n, where n is the number of cells being averaged [Rodriguez et al., Wilson et al., Sanders [Currently, the Shuttle Radar Topography Mission (SRTM) [r et al. ), and vo Sanders  showed tBrown et al., Lefsky, Simard et al., Baugh et al., One further problem that has received significant attention in recent years is the difficulty of obtaining a bare‐earth DEM from SRTM in vegetated areas. The inability of SAR to fully penetrate the tree canopy results in significant positive biases in the SRTM ground elevation over densely forested regions [1.2Lehner et al., Baugh et al., Simard et al., Elvidge et al., Gallant, Chase et al., The void‐filled Hydrosheds variant of SRTM is used as the base data set for this model [22.1Bergstrom and Forsman, Abbott et al., Huffman et al., Joyce et al., Dee et al., Xie et al., Kidd et al., Chen et al., Cohen Liechti et al., He et al., Chen et al., Gao and Liu, Sampson et al. [Blöschl et al., There are two main approaches to estimating extreme flood generating river discharge, namely from continuous simulation using hydrological models and flood frequency analysis. Hydrological models transform an input precipitation time series into an output river discharge time series and range in complexity from simple spatially lumped models such as HBV [n et al.  evaluateGaume et al., Merz and Blöschl, Zaman et al., Farquharson et al., Meigh et al., Merz and Blöschl, Smith et al., Pappenberger et al., Cameron et al., The alternative approach is to develop a regionalized flood frequency analysis (RFFA). Although differing in their specific methods, regional flood frequency analyses work on the assumption that data from data‐rich regions can be transferred to data poor ones. More specifically, these methods assume that catchments with similar characteristics will also exhibit similar flood frequency statistics. RFFA are well known in the literature and have been widely used to provide estimates of discharge in data poor regions [2.2As previously indicated, globally consistent observational data sets that allow simplistic catchment characteristics, such as area and rainfall, to be defined are now emerging. Although, the GRDC database has limited coverage in some regions, it contains sufficient quantity of data from all major climatic zones. Combined, these data sets enable RFFA to be applied at the global scale and subsequently allow preliminary discharge estimation with global coverage.Smith et al. [Kottek et al., k‐means clustering [Ramachandra Rao and Srinivas, Meigh et al., Zaman et al., http://nwis.waterdata.usgs.gov/nwis). Although the ability of this approach to provide detailed, localized discharge estimates is limited by the simplicity of the methods, significant uncertainties in the discharge record and the complexity of anthropogenically modified river systems, these methods have demonstrated skill in providing first‐order discharge estimates in data poor regions [Padi et al., Smith et al., Smith et al. [The method used here follows that described by h et al.  using a h et al. , far larThe above methods are based upon annual maxima (AMAX) data, and therefore enable AMAX discharge with an associated recurrence interval to be estimated. However, they do not constitute flood hydrographs that are required in order to enable hydrodynamic simulations to be undertaken. A simple design hydrograph is therefore generated using the rational hydrograph method, where the time to concentration for each catchment is calculated using Manning's equation to estimate routing velocity along the river network.2), an alternative method is required, with flow generated by raining directly on to the DEM (“rain‐on‐grid”). Therefore, in addition to the estimation of discharge, methods were also required for the estimation of extreme rainfall. This was achieved via a similar approach to the RFFA, only Intensity‐Duration‐Frequency (IDF) relationships were used as opposed to discharge data. IDF relationships are a standard engineering‐based method of estimating the intensity, duration, and frequency of extreme rainfall events. In total, IDF data from ∼200 locations were used. The procedure is as follows: IDF data from various locations around the world were pooled together and again partitioned into the various categories of the Koppen‐Geiger climate classifications [Kottek et al., Although the RFFA provides estimates of return period discharge for rivers and streams, it does not allow for accurate discharge estimation in small channels in which flooding is generally driven by intense local precipitation. For small channels  is likely to dominate boundary condition uncertainties (such as precipitation intensity) within the hydraulic model. Under such conditions, more accurate extreme rainfall estimates may not yield improvements in simulated flood hazard due to the DEM noise, and we therefore consider the method appropriate for this application.33.1Neal et al. [Lehner et al., Gong et al., Li et al., Yamazaki et al., As described by l et al. , the incLeopold and Maddock [Andreadis et al., Gleason and Smith, Miller et al., O'Loughlin et al., Pavelsky and Smith, Yamazaki et al., Recently, a number of studies have capitalized on the availability of remotely sensed data to generate regional to global estimates of river widths, either by building on the classical geomorphological relationships of  Maddock  at large3.2Fewtrell et al., In order to simulate extreme flows in river channels, it is necessary to estimate the conveyance capacity of the river, characterized by the river width and depth, as an accurate representation of channel bankfull capacity is necessary for behavioral simulations of flood events [Andreadis et al., Harman et al., Leopold, River depth is the most difficult parameter to estimate as it is not yet possible to measure this remotely on large scales. Where detailed local data are not available, it is necessary to infer an appropriate estimate of river depth at any location on the river network from available data. By assuming a bankfull discharge return period of approximately 1 in 2 years [The final stage is to decompose the model domain into individual reaches for simulation. Reaches are not arbitrary in length but are generated automatically according to a threshold difference in the modeled mean annual flood (MAF) between the upstream and downstream boundaries of the reach. Starting at the downstream end, the model tracks up the river network until the point is reached at which the MAF falls a threshold percentage below the downstream MAF . The RFFA is then used to generate a hydrograph for the downstream cell of the reach; the hydrograph is distributed among the upstream cell and any incoming tributaries according to their respective upstream catchment areas to ensure that the volume flowing into the reach at any point matches that of the RFFA‐generated downstream boundary. Each reach is simulated independently to prevent autocorrelation issues in the lower reaches of catchments.44.1Hallegatte et al., Brandimarte and Di Baldassarre, te Linde et al., Wesselink et al., Feyen et al., Elvidge et al., Jongman et al., If the global flood hazard model is to be used for flood risk estimation where more detailed resources are not available, then it is necessary to consider the impact of flood defenses as the majority of risk will be situated in urban areas where the potential for damage (“exposure”) and defenses (“mitigation”) is highest. Representing flood defenses in a global flood model is challenging because the engineered features themselves will typically be far smaller than the model grid scale and little or no data will be available to characterize them in most locations; the latter limitation also applies to the operating rules of reservoirs that are often designed to mediate fluvial flood risk. However, it is a fair assumption that most urban areas situated on floodplains will have some degree of flood defense, and that the level of defense will be related to the wealth and development of the country in which it is located. Literature relating to global flood defenses is scarce; a recent World Bank led study attempted to characterize defense levels in major coastal cities [4.2The benchmark data used in this study assume that defenses have failed, and so a defense method is not implemented here. However, it is possible to incorporate defenses within the framework by increasing channel conveyance according to an estimate of local defense standards. A method for estimating these standards based on socioeconomic factors and remotely sensed urbanization data sets will receive full treatment in a future study.55.1Bates et al., Hunter et al., Kalyanapu et al., Lamb et al., Neal et al., Yu, The emergence of highly efficient algorithms to describe the flow of water over the land surface in two dimensions has been pivotal in enabling the development of large‐scale hydraulic models. A novel simplified implementation of the shallow water equations [Neal et al. [One important limitation of standard 2‐D approaches over large domains is the inability to represent rivers whose width is considerably smaller than the grid size. For global models, where grid scales may be limited by terrain data resolution, subgrid methods and hybrid 1‐D/2‐D models have emerged as potential methods for representing such channels. l et al.  tested fAckerman et al., An unavoidable complication of a global model is the range of topographies that will be encountered. The types of model used to simulate inundation are typically derived from the shallow water equations, themselves a simplification of the Navier‐Stokes equations made under the assumption of large horizontal length scale relative to vertical length scale, implying a small vertical velocity. This assumption is reasonable on most floodplains where the gradients are low, but in order to ensure model stability and conservation of mass it is necessary for the model to be able to handle the areas of steep or discontinuous terrain that will inevitably be encountered in a high‐resolution global model. Within channel, it is possible to represent inline nonshallow water features such as waterfalls and control structures using the empirical weir equation [5.2Bates et al., Neal et al., Sampson et al., Kent et al., The hydraulic engine used here is based on the subgrid variant of LISFLOOD‐FP and employs the efficient inertial formulation of the shallow water equations [Schumann et al., Inundation dynamics on large rivers are simulated at 30 arc sec resolution rather than 3 arc sec resolution as DEM noise is reduced on the coarser grid, enabling a more stable estimate of water surface elevation to be produced over large floodplains. This has the additional advantage of reducing model runtimes by more than 2 orders of magnitude, allowing the long inundation events associated with seasonal floods on some major rivers to be simulated fully. A smooth water surface is then calculated by interpolating between points at the center of each 30 arc sec cell, enabling water heights to be reprojected onto the 3 arc sec DEM to obtain simulated water depths at the higher resolution. The decision to preserve water surface elevation, rather than mass, when downscaling was taken due to the large vertical noise error of the 3 arc sec SRTM DEM relative to the 30 arc sec SRTM DEM. A previous study has shown that downscaling flood extents onto a noisy DEM using a mass‐conservative method can lead to large local errors, and that under such conditions it may instead be preferable to preserve the water surface elevation simulated on the coarse, low noise, DEM while allowing some local extension to the downscaled flood extent where appropriate according to the high‐resolution topography [66.1The complexity and sheer size of a high‐resolution global flood model require that most elements of the model build process be automated. We developed a modular near‐automated model build framework augmented with python‐based GIS routines to generate suitable input data. The framework implements the methods outlined in the sections above and controls all stages of the hydraulic model build, execution, and postprocessing. A flowchart for the framework is provided in Figure 2The globally applicable model used here produces 3 arc second (∼90 m) resolution maximum water depth maps across the entire simulated global domain for 10 specified return period flows. The automation framework constructs tiles that are executed independently and then merged to form continuous data layers; the tiles have a 1° overlap across which a weighted blend is calculated to reduce the impact of anomalous boundary effects at the edge of each simulation domain. The total runtime for a typical 10° × 10° equates to approximately 2000 h on a single core of a 3.2 GHz Intel i7 processor, although the exact value for each tile varies according to several factors, including percentage land surface, climate , and hydrograph durations on the largest rivers in the tile. As LISFLOOD‐FP is Open‐MP parallelized, and the reach decomposition approach enables multiple simulations to be run simultaneously, model runtime can be shortened significantly where sufficient computational power is available: it is typically possible to build, execute, and postprocess a single 10° × 10° tile in under 24 h using a 200 processor core cluster. An example of raw model output for a 1 in 100 year return period simulation across Africa is shown in Figure A more thorough validation of the performance of flood hazard maps is challenging as they do not attempt to recreate the characteristics of a single observable event, instead highlighting those areas at risk from plausible events of a given type and magnitude. A pragmatic approach to validating the results of a global model is to compare the results against an analogous regional data set constructed using high‐resolution models and detailed local data. This approach has precedence and was recently used to assess the performance of the aforementioned EC‐JRC model; we employ it here using local hazard maps from Canada and the United Kingdom as our benchmark data sets. Results from the United Kingdom are also compared directly with results from the EC‐JRC model. To the author's best knowledge, this validation is more substantive than any other yet published for a global flood hazard model, but it is still subject to some clear limitations in regard to the size and location of the test sites. Ideally, the model would be validated against a high‐resolution flood hazard layer from a large catchment in a data‐scarce region, but by definition such a data set does not exist as it would require a large amount of detailed data to produce. Our validation therefore focuses on catchments where data availability has allowed the creation of high‐quality benchmark flood hazard information , but it should be noted that no such local data  were used to calibrate or otherwise modify the standard build procedure of the global model in the results presented below.2.1Alfieri et al., Aronica et al., Bates and De Roo, Fewtrell et al., Werner et al., H), sometimes referred to as the probability of detection, is a simple measure that indicates how well the model replicates the benchmark data without penalizing for overprediction:mA is the modeled inundated area and bA in the benchmark inundated area. H ranges from 0 to 1, with a score of 1 indicating that all wet cells in the benchmark data are wet in the model data. The False Alarm Ratio (F) is a measure of model overprediction :C) extends on H and F to create a combined score that penalizes for both underprediction and overprediction:B) is used:B varies between 0 and 1 indicates a tendency of the model to underpredict and a bias between 1 and ∞ indicates a tendency of the model to overpredict.The performance measures used are spatial variants of the commonly employed Hit Rate, False Alarm Ratio, and Critical Success Index, the latter of which is often referred to within the hydraulic modeling community as the “Fit” statistic [Results from the 3 arc‐second model runs were converted from their native raster format to a binary wet/dry shapefile format to allow the above metrics to be calculated. This was done in order to avoid resampling the higher resolution benchmark data to the lower model grid resolution.AE) between aggregated and benchmark data is then calculated:M is modeled flooded fraction, B is benchmark flooded fraction, and N is number of cells. The error for each catchment is calculated under two conditions: (i) exclusive of cells that are dry in both benchmark and model and (ii) inclusive of cells that are dry in both benchmark and model. An aggregate error bias (AB) metric is also calculated:In addition, aggregate performance metrics were also used. Many end users of global hazard models aggregate the data to a level that is commensurate with their operating scale. For example, metadata for insurance policies in the developing world are often poor with limited (or no) geocoding, resulting in considerable uncertainty about the physical location of an asset; commonly all that will be known is a high‐level postcode or administrative unit such as county or district. To give an indication of model performance under these conditions, the binary benchmark and simulated data for the catchment‐wide test cases are aggregated to a 30 arc sec (∼1 km) grid, with each pixel taking a value between 0 and 1 to represent the fraction of the cell flooded. The mean absolute error , each of which has a river running through it . These three rivers have their origins in the Rocky Mountains and flow through postglacial valleys on gravel beds. Flood hazard along these rivers is most severe in spring and summer when alpine snow melt can combine with spring rainfall or convectional summer storms to produce extreme discharge events. The data set was produced as part of the Alberta Flood Hazard Identification Programme by simulating 1 in 100 year design floods using steady state 1‐D Hydrologic Engineering Center River Analysis System (HEC‐RAS) models. The 1‐D water levels were reprojected onto the DEM using an AutoCAD/QuickSurf software package to create the 2‐D flood outline. The models were built using ground surveyed channel cross sections (including defenses) in conjunction with 1:5000 scale 1 m contour maps; design flows were derived from federal flow records from the Water Survey of Canada (part of Environment Canada). The Alberta data set has limited coverage as HEC‐RAS models were only built for certain river reaches and we therefore limit our analysis to the corresponding domain areas of the benchmark data. It was also necessary to mask a number of minor channels present in the global model but not in the benchmark data to ensure a fair comparison. This was generally straightforward as only major tributaries were modeled within the benchmark data; where minor tributaries were clearly missing from the benchmark data the corresponding channels were removed from the global model output.2, respectively) and were supplied by the Environment Agency of England and Wales as shapefiles. The data are multisource and are composed, in descending order of preference, of (a) flood extent observations of events with an estimated return period of ∼100 years; (b) detailed 1‐D or 1‐D/2‐D hydraulic models build using airborne LIDAR data with a spatial resolution of ∼2 m and a vertical accuracy of <10 cm; (c) a 2‐D hydraulic model constructed using airborne interferometric synthetic aperture radar (IfSAR) terrain data with a spatial resolution of ∼5 m and vertical accuracy of ∼0.5–1 m. Flows for the hydraulic models were derived using flood frequency analysis methods based on extensive flow records [Institute of Hydrology, 2 as this constraint was imposed by the EC‐JRC model [Alfieri et al., The data from the United Kingdom comprise catchment‐wide 1 in 100 year flood outlines for the Severn and Thames catchments  slight differences in the 500 km2 mask and (ii) regridding of the benchmark data onto a 90 m raster grid in the earlier study.The performance metrics for the Thames and Severn catchments are presented in Table It is apparent from Table C scores of 0.56 and 0.67 for the Thames and Severn, respectively. To put these score into context, previous (50 m) spatial resolution event‐based modeling studies of smaller rural reaches of the Thames and Severn achieved maximum C scores against remotely sensed observed extents of 0.72 and 0.65, respectively [Aronica et al., Horritt and Bates, C against SAR‐derived observed extents of between 0.72 and 0.89 depending on the date of comparison [Bates et al., Fewtrell et al., Sampson et al., Yu and Lane, C, H remains quite high indicating that the model is indicating flood hazard in broadly the correct areas and that model output may still be valuable for applications where a higher F is acceptable.Both models exhibit relatively good skill on larger channels, with H, but this is because the models are severely overestimating flood extents as demonstrated by lower C scores, high F scores, and large positive error biases. This overprediction occurs because (i) noise or lack of resolution can cause flow blockages in the DEM that result in elevated water levels and increased flood extent and (ii) water velocities are low due to the lack of deep channels, again resulting in elevated water levels. Both of these problems are addressed by the subgrid scheme in which faster in‐channel flow is simulated and local topographic errors can be readily overcome by forcing monotonically decreasing channel bed elevations.Comparison of the full complexity global model relative to the simpler 2‐D only global model demonstrates a critical limitation of the simpler approach. The 2‐D only models consistently produces a higher 2, the subgrid‐enabled global model provides the closest match to the benchmark data. The other two models are both 2‐D only and yield similar C scores on the Severn catchment, with the EC‐JRC model having a lower overprediction bias than the simplified 2‐D only global model; these differences are likely to be due to the different methods used by the two models to generate hydrographs. However, on the Thames, the EC‐JRC model is significantly outperformed by both variants of the global model. As noted in the original analysis of the EC‐JRC model, the challenge of using a hydrological model to derive extreme event hydrographs for the complex chalk‐based Thames catchment may offer a partial explanation. However, some of the underprediction exhibited by the EC‐JRC model within London is also due to the urban elevation bias of SRTM, something that is alleviated by the simple SRTM urban filter used by the global model presented here.Of the three model variants that only consider channels with an upstream area of >500 kmModel performance when aggregated to a ∼1 km grid is strong, and the small aggregate errors show a similar pattern between the model structures, with the full complexity global model outperforming the 2‐D only variants that again show a tendency toward overprediction. However, the difference between small and large catchments is not evident; this is because for minor channels, the area of flooding relative to the total pixel area is small, leading to a small aggregate errors. Furthermore, where the pixel‐by‐pixel comparison penalizes imperfect alignment in flooded areas caused by errors in the SRTM terrain data, the aggregate comparison only penalizes if the offset is large .3Chen et al., This paper presents a schematic framework for constructing a high‐resolution (3 arc sec or ∼90 m) global flood model by leveraging a number of recent advances in remote sensing and hydrology. A key constraint when developing the framework was to ensure that all data sets required for its construction had near‐global coverage as many of those traditionally required to build and calibrate flood hazard models  are relatively scarce outside western Europe and North America  [The setup presented here differs from previously published global flood models in that it attempts to model inundation dynamics using a two‐dimensional hydraulic model operating at resolutions commensurate with the highest quality existing global terrain data sets. Its closest existing published counterpart is the pan‐European EC‐JRC model, but two key differences exist between the two approaches. The first of these is that the method presented here challenges the assumption that a hydrological model is required to drive a large‐scale hydraulic model due to the lack of suitable data for calibration and validation globally, instead opting to apply a regionalized global flood frequency analysis. Although a flood frequency approach has been used, this study does not make any comparison between the methods used to derive discharge for global scale models; such a comparison is beyond the scope of this study. Work comparing the performance of these methods across data‐rich and data poor regions represents an area of research that needs to be undertaken in the near future. However, the application of a flood frequency analysis presents an advantage in that estimates of discharge can be easily coupled to channel size.Winsemius et al., Yamazaki et al., Alfieri et al., Neal et al., This links to the second key difference which is the explicit inclusion of a detailed river channel network, parameterization of which is tied to the flood frequency analysis to ensure that discharge estimates are commensurate with channel geometry estimates at any given point on the network. This is unique as previous studies at continental or global scales have either used 1‐D routing schemes with a subsequent postprocessing approach to derive flood depths or extents [Reuter et al., Rexer and Hirt, Schumann et al., Durand et al., Hinkel et al., Lewis et al., The model framework described in this paper is presented as a “first generation” global flood hazard model and as such is subject to a number of uncertainties and limitations that should be addressed by future work. In our opinion, the dominant data uncertainty in such a model remains the quality of terrain data available for use. The shortcomings of the SRTM data set are well documented [e.g., By considering both the performance of the model and the known limitations outlined above, it is possible to comment on the applicability of the model in its present form. The model has been demonstrated to perform well for inland flooding on smaller rivers than those previously associated with global flood hazard models , and the terrain correction applied to SRTM also enables the model to simulate flood hazard on vegetated and urbanized floodplains. However, the model is likely to underestimate flood hazard in coastal areas that are prone to tidal flooding due to the lack of a storm surge component. The model may also overestimate hazard along rivers with significant reservoir management capacity as such features are not yet incorporated. Finally, the effect on the model of extremely high infiltration rates in some arid and semiarid areas is as yet unknown due to a lack of suitable validation data, and therefore model results from these regions should be interpreted with care.4Validation of a flood hazard map is challenging as such data do not attempt to describe any single real event but instead attempt to describe the areas affected by all events of a certain magnitude. Previous global flood models have operated at resolutions that are too coarse to allow meaningful comparison to high‐quality local or national data, but the model presented here is sufficiently resolved to enable this approach. We present quantitative metrics that describe the global model's ability to replicate the flood extents shown by benchmark data sets from Canada and the UK. These metrics indicate that the global model is typically able to capture between two thirds and three quarters of flooded area in the local benchmark data, and that along nontidal reaches of large rivers, the skill is likely close to that of local models. Perhaps surprisingly, the model produced similar performance metrics in Canadian urban test cases as a result of the preprocessing of the underlying SRTM terrain data to reduce the elevation biases induced by radar returns from the tops of buildings. Skill relative to the benchmark data sets is observed to decrease along smaller channels as more subtle topographical features are unresolved in SRTM data and noise in the DEM can often lead to errors in the location of streams and minor tributary rivers within the model. The general smoothing of small topographical features also offers an explanation for the tendency of model errors to be biased toward overprediction.Many practical applications of global hazard data sets such as this involve working at relatively coarse scales in conjunction with other global data sets that may have considerable spatial uncertainties. As it is common practice to aggregate hazard data to a scale that is commensurate with the operating scale of the application, the performance of the model when aggregated to a ∼1 km grid was also calculated for the UK whole‐catchment test cases. Under these conditions, model performance was strong, with mean absolute error in flooded fraction typically below 5% across both the Thames and Severn relative to similarly aggregated benchmark data. The aggregate performance indicates potential for this type of model to be used for preliminary flood hazard assessment in areas where more detailed local data are not available. However, at smaller scales, topography remains the single largest limiting factor in models of this type, and the forthcoming release of new higher resolution terrain sets, such as global public release of 1 arc sec SRTM and the TanDEM‐X Elevation12 DEM, offer exciting prospects for improvement in the near future.ErratumIn the originally published version of this article, author Paul D. Bates's middle initial was incorrect. This has since been corrected and this version may be considered the authoritative version of record.


To accelerate progress toward the Millennium Development Goal 4, reliable information on causes of child mortality is critical. With more national verbal autopsy (VA) studies becoming available, how to improve consistency of national VA derived child causes of death should be considered for the purpose of global comparison. We aimed to adapt a standardized computer algorithm to re–analyze national child VA studies conducted in Uganda, Rwanda and Ghana recently, and compare our results with those derived from physician review to explore issues surrounding the application of the standardized algorithm in place of physician review.We adapted the standardized computer algorithm considering the disease profile in Uganda, Rwanda and Ghana. We then derived cause–specific mortality fractions applying the adapted algorithm and compared the results with those ascertained by physician review by examining the individual– and population–level agreement. Our results showed that the leading causes of child mortality in Uganda, Rwanda and Ghana were pneumonia (16.5–21.1%) and malaria (16.8–25.6%) among children below five years and intrapartum–related complications (6.4–10.7%) and preterm birth complications (4.5–6.3%) among neonates. The individual level agreement was poor to substantial across causes (kappa statistics: –0.03 to 0.83), with moderate to substantial agreement observed for injury, congenital malformation, preterm birth complications, malaria and measles. At the population level, despite fairly different cause–specific mortality fractions, the ranking of the leading causes was largely similar.The standardized computer algorithm produced internally consistent distribution of causes of child mortality. The results were also qualitatively comparable to those based on physician review from the perspective of public health policy. The standardized computer algorithm has the advantage of requiring minimal resources from the health care system and represents a promising way to re–analyze national or sub-national VA studies in place of physician review for the purpose of global comparison. To accelerate progress toward the Millennium Development Goal 4 (MDG 4)  in 2015 VA studies by design should be consistent and comparable when used to generate population level cause–of–death estimates . In pracCompared to computer algorithm, physician review has the apparent disadvantage of involving a large team of physicians. This may intervene with the routine function of the health care systems in many LMICs. In addition, physician review derived cause–of–death estimates may have limited internal consistency due to concerns over repeatability of the approach ,14,15,17In this study, we aim to adapt a previously developed standardized computer algorithm  to re–anThe study data were publically accessible and analyzed anonymously. Hence no informed consent was needed.The three national Child VA studies re–analyzed includes the 2007 Uganda study, 2008 Rwanda study, and 2008 Ghana study ,15. The th revision (ICD–10). If discordant ICD–10 codes were assigned by the two coding physicians, a single cause of death was agreed upon after deliberation.All three VA studies followed the Sample Vital Registration with Verbal Autopsy (SAVVY) protocol . SpecifiWe did not have access to the open–ended narrative data and focused the current analyses on the structured section of the VA questionnaires. We applied the following criteria to only include children who: 1) were identified as live births; 2) died between ages 0 and 59 months; 3) had complete VA interviews; 4) had sufficient information on age at death; and 5) were administered age–appropriate questionnaire.Online Supplementary Document(Online Supplementary Document)). Generally, the algorithm only allowed one cause for each death. But deaths due to measles, diarrhea and acute respiratory infection (ARI) can be assigned simultaneously, and then re–distributed into the respective single cause according to their cause–specific mortality fractions (CSMFs) assigned before the re–distribution. Causes were assigned through a hierarchical process in which diagnoses with more specific symptoms were made before those with less specific ones, and cases without any diagnosis at the end were classified as unspecified conditions  –31, we dOnline Supplementary Document(Online Supplementary Document).We also added meningitis/encephalitis and neonatal sepsis in the algorithm. We adopted case definitions of the two conditions from WHO , and plaOnline Supplementary Document(Online Supplementary Document) maps the ICD–10 codes and the cause categories used in the standardized computer algorithm and physician review.We grouped causes of deaths into categories comparable to the Child Health Epidemiology Reference Group (CHERG) categorization, including among neonates: pneumonia, preterm birth complications, intrapartum–related complications (including birth asphyxia and birth injury) , sepsis,To compare results derived from standardized computer algorithm vs physician review, individual–level concordance within each country was examined using Cohen’s kappa, where deaths initially assigned to multiple causes  were excluded. Population level agreement was also assessed by comparing CSMFs and ranking of the CSMFs of the top five single causes . All anaAmong the 724, 462 and 226 deaths available in the child VA studies in Uganda, Rwanda and Ghana, 530 , 360  and 188  deaths met the study inclusion criteria, respectively.The CSMFs among neonates, children aged 1–59 months and 0–59 months estimated applying the standardized computer algorithm are presented in Individual–level concordance between causes assigned by the two approaches is shown in CSMFs derived from the standardized computer algorithm in comparison with those from physician review among neonates and children aged 1–59 months are presented in The ranking of the top five single causes is show in In this paper, we derived comparable causes of child mortality distributions by applying an adapted standardized computer algorithm to re–analyze national VA data collected in Uganda, Rwanda and Ghana in 2007–2008. Overall, the distribution of child mortality is by and large similar across the three countries based on our results, with malaria and pneumonia being the leading causes of under–five deaths and intrapartum–related complications and preterm birth complications being the major neonatal causes.However, the distribution in Ghana appeared to be different from that in the other two countries in that the diarrhea–specific mortality fraction was much smaller, the AIDS–specific mortality fraction was larger, and more deaths were not assigned. We speculated that the differences between Ghana and the other two countries may be partially attributable to the measurement and data quality issues in the Ghana VA study. Only 2.5% of under–five deaths were assigned to diarrhea in Ghana, which was implausibly low compared to existing studies ,39. A clThe kappa statistics showed substantial to poor agreement between results generated by the two ascertaining methods, suggesting that the individual–level agreement varied greatly by cause. Causes with more distinct signs and symptoms, such as injury, congenital malformation, preterm birth complications, and measles had a higher level of agreement compared to other causes. Malaria also had moderate agreement, which may be associated with its high prevalence. Other infectious causes like pneumonia, diarrhea, sepsis and AIDS all have fair to poor agreement, probably due to their non–specific symptoms and likely comorbidity with other infectious conditions. It is noted that the low specificity associated with infectious causes is not unique to computer algorithm, rather, it is a common issue shared by all methods ascertaining causes of death ,42. PoorThe absolute values of CSMFs assigned by the standardized computer algorithm were fairly different from those assigned by physician review among neonates and children aged 1–59 months. However, when ranking of the leading five causes and their specific ranks were compared, many similarities can be drawn between results derived from the two approaches. It suggests that public health policy decisions could be largely similar based on distribution of causes of child mortality derived from both methods .The study has several limitations. First, our preliminary child AIDS case definition has not been validated. The fact that the AIDS–specific fraction did not have a linear relationship with the adult HIV prevalence in the three countries could also suggest that our AIDS case definition have unsatisfactory validity. However, more specific signs and symptoms are generally required to improve the validity of the AIDS case definition. Additional efforts are urgently needed to develop and standardize AIDS case definition among children that can be used in national VA studies.Second, the standardized computer algorithm assigned 38% of post–neonatal deaths to malaria in Rwanda, which is unlikely to be plausible. CHERG estimated that 3.7% of post–neonatal deaths were attributable to malaria in Rwanda in 2008 . AnotherIn addition, neither the standardized computer algorithm nor physician review is capable of providing the “true” causes of death. Both approaches could be equally invalid. When the two approaches agree, it does not necessarily mean that there is greater truth to the causes assigned.Despite these caveats, it is feasible to re–analyze national VA studies applying a standardized computer algorithm for the purposes of cross–country comparison and global burden of childhood disease estimation. The standardized computer algorithm produced internally consistent and comparable distribution of causes of child mortality in comparison to physician review. It also has the advantage of requiring minimal resources from the health care system. From the public health policy stand point of view, the standardized computer algorithm and physician review also generate similar sets of leading causes of child deaths. The standardized computer algorithm represents a promising way to re–analyze national or sub–national VA studies in place of physician review. It could be further strengthened with improved validity of child AIDS case definitions. The standardized computer algorithm should be of particular importance in sub–Saharan Africa, where human capital and financial shortfalls are the greatest. The application of a standardized computer algorithm on child VA data are one step forward toward the harmonization of cause–of–death reporting and estimation in children younger than five years. Among the CHERG community, discussion is on–going about how to utilize national VA data so that consistent and comparable cause–of–death estimates can be generated across countries and time for the purpose of global burden of childhood disease estimation. We welcome a discussion on this subject among a wider community.


Proteins and bacteria on the skin of amphibians can protect them from diseases. Marking frogs with elastomer tags had no effect on stress hormone production or protein release, but lead to a slightly greater abundance of skin microbes. This marking technique may be a comparatively safe option for amphibians. Batrachochytrium dendrobatidis. Events that cause disruption of symbiotic bacterial communities or deplete peptide stores could increase the susceptibility of individuals to disease and may have implications for amphibians involved in probiotic trials or time course studies that investigate symbiotic bacterial communities. It has previously been shown that passive integrated transponder tagging of frogs causes a rapid (within 24 h) and major proliferation of micro-organisms on the skin. Here, we show that marking of red-eyed tree frogs  with visible elastomer has no effect on adrenal response  or peptide production, although there was evidence of a slightly greater microbial abundance associated with the skin of marked frogs 2 weeks after tagging. The results indicate that visible elastomer may be a preferable marking technique to passive integrated transponder tagging, particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. More work is required to determine the effects of different marking techniques on physiological responses of amphibians, whether these physiological responses are consistent across host species and whether such ‘non-invasive’ marking methods affect the susceptibility of amphibians to infectious pathogens, such as B. dendrobatidis.Amphibians possess innate immune defences, including antimicrobial peptides and symbiotic bacterial communities, that can protect them from infectious diseases, including chytridiomycosis. On-going research is attempting to use amphibian symbiotic bacteria to develop probiotic treatments that can protect hosts from the causative agent of chytridiomycosis, the fungal pathogen  Batrachochytrium dendrobatidis, from a number of amphibian species  tagging or visible implant elastomer (VIE) dyes, are now more commonly used. The existing literature indicates that these marking techniques have few negative effects on amphibians  marked with VIE or toe-clipped under varying degrees of anaesthesia did not exhibit increased noradrenalineor adrenaline levels in comparison to unmarked salamanders. Handling has been shown to increase circulating glucocorticoid concentrations in a range of amphibian species .There are limited studies that have investigated the effects of marking techniques on steroid  production in amphibians.  species , 2013, aThe Chester Zoo Ethical Committee and the University of Manchester Ethics Committee approved this study prior to starting. This study did not require a UK Home Office licence because VIE tagging is an approved method for the identification of frogs, and data were collected during routine marking of frogs for identification purposes. Frogs were checked daily throughout the study and for 2 weeks after for any signs of adverse reaction to any of the techniques used, none of which was observed.A. callidryas were used in this study; eight  in the control (unmarked) group and eight  in the marked group. All frogs were from the same clutch of F3 generation captive-bred individuals and had not previously been involved in any other studies. Prior to the start of the experiment, frogs were group housed in 60 × 45 × 45 cm glass tanks  with a naturalistic set-up consisting of a LECA base for drainage and a soil layer covered with dried oak leaves, and planted with a peace lily and cuttings of devil's ivy. Two weeks prior to the start of the study, frogs were randomly assigned to 16 individual experimental housing tubs (30 × 18 × 20 cm ExoTerra® plastic faunariums), with frogs from the same group housed tanks spread across treatment groups to allow for prior variation in bacterial communities. Individual housing was required to distinguish faecal samples between individuals and to avoid the potential for bacterial communities to be confounded by group housing during the study. Experimental tanks were lined with damp paper towels and contained a water dish and a cutting of devil's ivy. Paper towels and water dishes were changed twice weekly. All tanks were maintained with a ZooMed Reptisun® 10.0 ultraviolet strip light with reflectors and a Philips daylight bulb with reflectors, both of which were on a 10:14 light:dark cycle. Frogs were fed three or four times weekly with black crickets (Gryllus bimaculatus) gut-loaded on fresh fruit and vegetables and dusted with Nutrobal .A total of 16 adult Faecal glucocorticoid metabolite concentrations’ section below for further details). On day 8, frogs were sampled for bacterial communities and peptide release as described below . This provided baseline data for each individual, which is referred to hereafter as ‘start of study’. On day 14, frogs in the ‘marked’ group were tagged using VIE dye (see ‘Marking techniques’ below for details). On day 15, all frogs were sampled again for bacterial communities and peptide release, and these data are referred to as ‘post-marking’. On day 28, bacterial communities and peptide release were sampled again, and these data are referred to as ‘end of study’.After a 2 week habituation period in the individual housing, faecal samples were collected daily from day 1 until the end of the study (day 28) to measure faecal glucocorticoid metabolite concentrations . This method was chosen because frogs were due to be rehomed at another institution and it was necessary to distinguish these frogs from existing A. callidryas in the collection at the receiving institution without the requirement for individual identification. The elastomer dye was prepared according to the manufacturer's instructions. Gloves were worn throughout marking and changed between frogs to minimize cross-contamination. During marking, the frog was restrained on a flat and stable surface that had previously been sterilized, and the sterile needle containing the dye was inserted smoothly and quickly under the skin of the underside of the tibiofibular . The marking process was standardized and took ∼30 s per individual. In order to determine whether the whole marking process affected physiological traits of frogs, control frogs were not sham injected or manipulated at the post-marking time point. All frogs were monitored for the remainder of the study and for 2 weeks after the end of the study for any signs of adverse reaction, none of which was observed.A. callidryas are nocturnal and so defaecate at night), and any faecal samples were frozen at −20°C for subsequent analyses. Gloves were used to handle samples and changed between to avoid contamination. Glucocorticoid metabolite analyses were conducted at the Chester Zoo's Wildlife Endocrinology Laboratory.Tanks were checked daily within an hour of the lights coming on , and 50 μl per well was loaded onto a 96-well Nunc-Immuno Maxisorp (Thermo-Fisher Scientific) microtiter plate, which was covered with a plate sealer and left overnight at 4°C. Plates were washed five times  and then 50 μl per well of each standard and sample (3.9–1000 pg per well or samples diluted 1:10 in EIA buffer) were loaded in duplicate with 50 μl per well of horseradish peroxidase conjugate diluted in EIA buffer (1:70 000). Following incubation in the dark for 2 h at room temperature, plates were washed five times and incubated with 100 μl per well of substrate , left to develop at room temperature in the dark and measured at 405 nm at optical density 0.8–1.0. The corticosterone EIA was biochemically validated for measuring corticosterone metabolites in A. callidryas faeces through parallelism  and matrix interference assessment . The cross-reactivities for corticosterone antiserum CJM006 have been reported elsewhere .Glucocorticoid metabolites (corticosterone) were analysed using a modified enzyme immunoassay (EIA) as previously described  and stanpost hoc analysis for pairwise comparisons.Final corticosterone metabolite concentrations were calculated per gram of wet faecal mass. Data collected before any handling or marking events days 1–7) were assigned as ‘start of study’, data collected from 1 day post-marking until 5 days post-marking (days 16–19) were assigned as ‘post-marking’, and data collected from day 20 until day 28 were assigned as ‘end of study’. Raw data values for faecal glucocorticoid metabolite concentrations indicated an increase at around 3–5 days post-marking . Frogs were then swabbed on their ventral surface 20 times using sterile swabs . Gloves were worn throughout the swabbing process, which took ∼30 s per frog, and changed between individuals to minimize cross-contamination. Swabbing methods were consistent across individuals, treatment groups and time points. Swabs were placed into 1 m NaCl2 solution and serial dilutions constructed up to a concentration of 10−2. Concentrations of 10−1 and 10−2 were plated out on low-nutrient agar . Bacterial and fungal colony abundances were counted every 2 days until negligible new growth was observed . Bacterial and fungal counts were then multiplied by their respective dilution factors and averaged for each individual. Data for bacterial abundance and fungal abundance were analysed separately in RStudio (using the lme4 package) for effects of treatment, time point and the interaction between treatment and time point using a generalized linear mixed model with Poisson distribution, with individual included as a random effect. Tukey's post hoc analysis was conducted for pairwise comparisons.Microbial communities were collected using similar methods to those previously described by Prior to sampling for cutaneous microbes, frogs were rinsed on their dorsal and ventral surfaces by pouring 50 ml of ultrapure water over each surface five times. The final volume of liquid collected was recorded, and peptides were extracted from the water using previously described techniques  according to the manufacturer's instructions, except that bradykinin  was used to establish a standard curve  but not treatment  or the interaction between treatment and time  and between control frogs post-marking and at the end of the study (P = 0.001), with end-of-study faecal glucocorticoid metabolite concentrations being lower at the end of the study than at the other two time points , and post hoc analyses showed that faecal glucocorticoid metabolite concentrations at the end of the study were significantly lower than those at the start of the study (P < 0.001) and post-marking (P < 0.001) across all frogs, but there were no significant differences between faecal glucocorticoid metabolite concentrations at the start of the study and post-marking (P = 0.377).There was a significant effect of time  and the interaction between treatment and time  on bacterial abundance, but not of treatment alone . Post hoc analyses showed overall that there were significant decreases in bacterial abundance over time for both treatment groups, and the bacterial abundance associated with control frogs at the end of the study was significantly lower than that of marked frogs at all time points  on the abundance of fungi associated with the skin of frogs, but no significant effects of treatment  or the interaction between time and treatment . Post hoc analyses showed that marked frogs had a significantly greater fungal abundance at the end of the study than post-marking , treatment  or the interaction between treatment and time  on the release of peptides by frogs .There were no significant effects of time , with frogs maintained individually exhibiting lower faecal glucocorticoid metabolite concentrations than those maintained in groups of two or eight, and those maintained in group housing exhibiting a decline in glucocorticoid metabolite concentrations over the 4 week study period.In this study, we found no evidence that VIE marking of frogs leads to an increase in faecal glucocorticoid metabolite concentrations in comparison to unmanipulated control animals. It has previously been shown that marking ng alone , b, 2013A. certus and A. glyphus, and adrenocorticotrophic hormone challenges in these species indicated a similar lag time in glucocorticoid metabolite deposition in the faeces as for A. callidryas in this study , indicating that these species may primarily deposit glucocorticoid metabolites in their faeces.Faecal samples have been used to measure glucocorticoid metabolite concentrations in a range of taxa . llidryas , fungi wB. dendrobatidis after tagging with VIE and other marking systems. Moreover, culturing methods are known to underestimate microbial diversity greatly  on the skin of marked frogs is unclear. The VIE tags were unlikely to be completely sterile due to the preparation methods, and the greater abundance of microbes may reflect a minor infection from the tags. Alternatively, it is possible that the act of marking caused a disruption in the dynamics of the bacterial communities that may have had delayed effects for the microbial community or elicited a physiological response in the frogs. For example, studies in rats and humans have shown increases in immunoglobulins, lymphocytes and macrophages in response to injection of silicone  than for PIT tagging (18 gauge or 1.27 mm), and the ‘foreign body’ inserted under the skin is much smaller; therefore, frogs undergoing PIT tagging may experience greater adrenal activity than those that are VIE tagged, leading to differences in the responses of the microbial community .The results presented here are in contrast to the results found by A. callidryas. This result is expected given that VIE marking also had no effect of faecal glucocorticoid metabolite concentrations, which have the potential to inhibit the production of new peptides for storage , particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. Although more work is required to determine differences between host species in response to different marking techniques, as well as the effects of marking on the susceptibility of amphibians to disease, these results together with those of Overall, we have shown that there is no effect of VIE marking on adrenal response  or peptide release of Conservation Physiology onlineSupplementary material is available at .This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) PhD studentship to R.E.A.


Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate , or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle  species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi,  Many insects are involved in symbiotic associations with microbes that provide nutrition crucial for insect survival  are some of the most ecologically and economically important forest insects and many are involved in tightly linked ectosymbioses with fungi  symbiosis is a powerful system to address questions of coevolution and codiversification in ectosymbioses, in general, and beetle–fungus symbioses, in particular. This symbiosis involves a beetle with two symbiotic fungal partners, Entomocorticium sp. B (Basidiomycota) and Ceratocystiopsis brevicomi (Ascomycota)  across most of its range. Genetic evidence indicates the beetle is actually two cryptic species that are geographically isolated on two subspecies of ponderosa pine , while haplotype C occurs exclusively in the southwestern United States .The western pine beetle is restricted to ponderosa pine  whether we could detect evidence of adaptation by the beetle to specific isolates of E. sp. B. We did this by rearing beetles from one cryptic species with their normal haplotype of E. sp. B , with a haplotype associated with the other cryptic beetle species , or with no fungi. We then characterized the effect of these three treatments on beetle development and fitness.The objectives of this study were to determine whether (1) mutualistic fungi, particularly Live western pine beetles were collected near Missoula MT  in June 2012 using Lindgren funnel traps baited with chemical attractants . All adult beetles were surface‐sterilized (30 sec in 70% EtOH) to remove potentially antagonistic fungi which can be carried externally and hamper laboratory rearing. Sex was then determined by the presence of the mycangial swelling on the pronotum of females and tubercles on the frons of males Wood . Males aLarvae void their guts prior to pupation but may still carry microbes including fungi on their exoskeletons. Therefore, we surface‐sterilized the pupae using a series of three short EtOH washes conducted over 3 days. Washes consisted of placing pupae for 10 sec in 70% EtOH, before quickly dipping them in distilled water and transferring them to a Petri dish. We then placed surface‐sterilized pupae into pseudo‐pupal chambers constructed from fresh ponderosa pine bark , all of which were negative for fungi. Methods used to isolate mycangial fungi from western pine beetles are detailed in Bracewell and Six .E. sp. B, they are genetically divergent; MI22 has been identified as haplotype A and RO10 as haplotype C  was originally isolated from a beetle collected near Missoula MT . The non‐natal isolate (RO10) was originally isolated from an individual of the other cryptic western pine beetle species collected near Ruidoso NM   in August 2012. The tree was first cut crosswise into 33‐cm sections. Then, each section was quartered lengthwise resulting in a total of 198 cmDue to slight differences in phloem thickness among sections which could influence total brood production, treatments  were randomly assigned to the tree sections. Seventeen tree sections (replicates) per treatment were assigned to each of the three treatments at the start of the experiment. To establish the fungus in a section, one 4‐mm‐diameter plug of agar was taken from an MEA plate containing either the natal or the non‐natal fungus. No fungus treatments received a plug of MEA. Each plug was smeared inside a hole drilled into the phloem layer at the base of the tree section. Trials conducted prior to our experiment indicated that 7 days was adequate for both the natal and the non‐natal fungus to establish in the tree phloem. Therefore, after inserting the agar plug, all tree sections were held for 7 days. Surface‐sterilized female/male pairs were then inserted into the same drill hole , and a piece of wire screen was fixed over the hole to prevent escape. Each tree section was then placed in a rearing container and monitored daily for emergence of offspring. Tree sections were maintained at room temperature (~21°C) under natural light conditions.KJ620521 and KJ620518).To assess the effect of a particular treatment on brood production and the resulting fitness of offspring, we recorded when each brood beetle emerged, their sex, and their pronotum width. Pronotum width is a proxy measure for overall beetle size  tests in the R multcomp package  and thus modeled using a quasi‐binomial distribution. For all models, adequate model fit was determined by evaluating the residual deviance.All analyses were conducted using the statistical package R v. 3.1.2  were considered for analyses. Criteria for inclusion in analysis were that parent gallery length was >0 cm (indicating successful pairing and tunneling by the parents).n = 3 natal, 11 no fungus, 2 non‐natal) were kept in their respective treatment category. We sequenced the ITS2‐LSU regions of three isolates from the mycangia of beetles from three replicates each of the natal fungus treatment and the non‐natal fungus treatment (only three replicates had fungi), and one isolate from the only replicate of the no fungus treatment that was positive for mycangial fungi. All of the isolates possessed DNA sequences identical to haplotype A, indicating that there were instances in the non‐natal and no fungus treatments where surface sterilization of the pupae was unsuccessful. After removing these replicates from further analyses, we were left with, 14, 9, and 12 replicates per treatment . For the natal fungus treatment, we recovered natal fungi from 73% of replicates . In contrast, we were unable to recover the non‐natal fungus from a single mycangium of brood females   = 56.48, P < 0.0001). All tree sections in the natal and non‐natal treatments showed evidence of E. sp. B growth in the phloem in both the parent gallery and the larval tunnels.A total of 742 brood adult beetles were recovered from rearing containers. A subset of brood females from each replicate (when present) were used to isolate fungi from their mycangia to confirm that experimentally manipulated fungi were successfully transferred to the gallery of the insect and subsequently acquired in brood adult mycangia. We isolated fungi from the mycangia of 1 to 14 beetles from all replicates that produced females and in total isolated fungi from 132 individuals . There was also no effect of fungal treatment  . Results from size comparisons of brood males were similar to that of females. There was no significant relationship between the size of male beetles and their development time  nor did fungal treatment affect adult size  .When comparing the relative size of brood from the natal and non‐natal treatments, and with respect to brood females, we did not find a relationship between adult size and development time . Fungal treatment  also did not significantly influence development time , and there was no interaction between the development time of either sex and fungal treatment .Development time of brood beetles was highly variable  days and non‐natal beetles = 168.15 (±23.68) days). There was no significant difference in development times of either sex  was capable of supporting development of beetles from Montana. However, our most striking finding was the inability of brood adult beetles from Montana to acquire the southwest isolate/haplotype in their mycangia. Our results suggest that western pine beetles are adapted to particular fungal partners and that fidelity may be enforced through mycangial selectivity.Many insects rely on mutualistic symbionts for nutritional supplementation  system, where there is a strong negative effect on beetles when they are reared without their mycangial fungi (Entomocorticium sp. A and C. ranaculosus). Barras  in our study suggest that both are equally capable of providing the nutritional requirements of the host beetle. We observed no differences between natal and non‐natal treatments with regard to total number of brood adults, their size, or their development time, despite the fact that the two fungal isolates are genetically divergent , our results do not suggest that the non‐natal fungus grew or sporulated differently than the natal fungus. Had the non‐natal fungus grew poorly or not sporulated, brood adults would have likely been smaller  (Six and Paine There are a number of species in the bark beetle genus How the beetle maintains these associations through time is not well understood, but our results indicate mycangia may play a key role enforcing specificity. Bark beetle mycangia have complex morphologies and contain glands which secrete substances thought to nurture fungi during transport (Happ et al. For insects involved in obligate mutualisms with microbes, maintaining associations with particular partners is crucial for the success and stability of the symbiosis. However, currently, we have only a rudimentary understanding of the mechanisms that underlie fidelity or evolutionary processes in ectosymbioses, including bark beetle–fungus symbioses. We have shown in other work that the western pine beetle symbiosis exhibits high fidelity (Bracewell and Six None declared.